Search

Two new compounds, a rearranged limonoid (harriforanoid) and a benzopyran (harriforanone) were isolated from the leaves and stems of Harrisonia perforata (Blanco) Merr., together with three known limonoids and fourteen known chromones. The structures of the compounds were elucidated using spectroscopic analysis. The experimental and calculated ECD spectra were used to characterize the absolute configuration of harriforanone. The cytotoxicity of each compound was assessed towards cholangiocarcinoma (KKU-100) and human liver cancer (HepG2) cell lines. However, none of these compounds exhibited any activity (IC50 > 50 μM).

Purpose: To explore the effect of alendronate on cell death and migration of cholangiocarcinoma (CCA). Methods: Migration and cell death of CCA cells were determined using sulforhodamine B (SRB), colony formation, wound healing, and gelatin zymography assays. The mechanism of action of alendronate was studied with reverse-transcriptase polymerase reaction (RT-PCR) for gene expression and by Western blotting analysis for protein expression. Results: Alendronate stimulated KKU-100 cell death in dose- and time-dependent manner, with low IC50 value, and significantly inhbited colony formation at doses of 5 - 100 µM. Moreover, alendronate at doses of 250 - 1000 µM significantly stimulated CCA apoptosis via reactive oxygen species (ROS) generation, and enhanced caspase 3 activity at a dose of 1000 µM. Moreover, at a dose of 250 µM, it significantly inhibited cell growth through induction of caspase 3 and p53, and reduction of protein expression levels of NF-ĸB. Furthemore, alendronate altered mevalonate (MVA) pathway via downregulation of Rac1 protein expression. In contrast, it significantly inhibited CCA cell migration, and reduced MMP 2 and MMP 9 levels at doses of 25 - 100 µM. Conclusion: Alendronate may be useful as a novel drug for prevention and chemotherapy of CCA.

Objective Cholangiocarcinoma (CCA) is a noxious malignancy of epithelium of the bile duct with a low response rate to chemotherapy. The epidermal growth factor receptor (EGFR) signaling pathway is implicated in the development of cancerous cells, especially CCA. In this study, we report detailed biological profiling of 13f identified from our earlier hit expansion studies. The aim of this work was to expand our understanding of 13f via more detailed investigations of its mechanism of action against KKU-100, KKU-452 and KKU-M156 CCA cells, as well as in comparison to the EGFR inhibitor Gefitinib and non-specific chemotherapeutic agents such as Cisplatin. Methods Inhibiting EGFR-Kinase, cytotoxicity, clonogenic assay, wound healing and apoptosis were performed. Levels of total expression of EGFR and EGFR phosphorylation proteins were detected. Results 13f was confirmed as an inhibitor of EGFR with an IC50 value against the tyrosine kinase of EGFR of 22 nM and IC50 values for 48 h incubation period were 1.3 ± 1.9, 1.5 ± 0.4 and 1.7 ± 1.1 µM of KKU-100, KKU-452 and KKU-M156, respectively through dose- and time-dependent induction of early apoptosis of CCA cells. The compound also suppressed the clonogenic ability of KKU-100 and KKU-M156 cells stronger than Gefitinib, while potently inhibiting EGF-stimulated CCA cell migratory activity in KKU-452 cells. It was observed that under normal conditions EGFR was activated in CCA cells. EGF-stimulated basal expression of EGFR in KKU-452 cells was suppressed following 13f treatment, which was significantly greater than that of the marketed EGFR inhibitor Gefitinib. Conclusion In summary, our study showed that 13f has potent anti-cancer activities including antiproliferation, clonogenic ability and migration through the modulation of EGFR signaling pathway in CCA for the first time. The compound represents an interesting starting point as a potential chemotherapeutic agent in ongoing efforts to improve response rate in CCA patients. .

Objective: To evaluate the potential therapeutic effect of Sang-Yod rice bran hydrolysates (SRH) and in combination with lisinopril against hypertension, endothelial dysfunction, vascular remodeling, and oxidative stress in rats with nitric oxide deficiency-induced hypertension. Methods: Hypertension was induced in male Sprague-Dawley rats by administration of a nitric oxide synthase inhibitor, Nω- nitro-L-arginine methyl ester (L-NAME) in drinking water for 6 weeks. Hypertensive rats were administered daily with SRH (500 mg/kg/day), lisinopril (1 mg/kg/day), or the combination of SRH and lisinopril by gastric lavage for the last 3 weeks of L-NAME treatment. Hemodynamic status, vascular reactivity to vasoactive agents, and vascular remodeling were assessed. Blood and aortic tissues were collected for measurements of oxidative stress markers, plasma angiotensin-converting enzyme (ACE) activity, plasma angiotensin II, and protein expression. Results: L-NAME induced remarkable hypertension and severe oxidative stress, and altered contents of smooth muscle cells, elastin, and collagen of the aortic wall. SRH or lisinopril alone reduced blood pressure, restored endothelial function, decreased plasma ACEs and angiotensin II levels, alleviated oxidant markers and glutathione redox status, and restored the vascular structure. The effects were associated with increased expression of endothelial nitric oxide synthase and decreased expression of gp91phox and AT1R expression. The combination of SRH and lisinopril was more effective than monotherapy. Conclusions: SRH alone or in combination with lisinopril exert an antihypertensive effect and improve endothelial function and vascular remodeling through reducing oxidative stress and suppressing elevated renin-angiotensin system.

Objective: To investigate anti-tumor effect of rice bran hydrolysates (RBH) on proliferation, migration, invasion, and angiogenesis of cholangiocarcinoma (CCA) cells, and elucidate the underlying mechanisms. Methods: RBH was prepared from Tubtim Chumprae rice (Oryza sativa L.) by hydrothermolysis followed by protease digestion. Phenolic content in RBH was analyzed by high-performance liquid chromatography. Human CCA cells, KKU-156, KKU-452, and KKU-100, were used to study the effects of RBH on proliferation, migration, invasion, and adhesion by wound healing, Transwell chamber, and fibronectin cell adhesion assays. Angiogenesis was evaluated using human umbilical vein endothelial cells. Proteins associated with cancer progression were analyzed by immunobloting assays. Results: RBH contained carbohydrates, proteins, lipids, and various phenolic compounds and flavonoids. RBH did not inhibit CCA proliferation, but strongly suppressed migration, invasion, adhesion of CCA cells, and the formation of tube-like capillary structures of human umbilical vein endothelial cells. Moreover, RBH down-regulated phosphorylation of FAK, PI3K, and Akt, suppressed NF-κB nuclear translocation, decreased the expression of ICAM-1, vimentin and vascular endothelium growth factor (VEGF), and increased the expression of E-cadherin. Conclusions: RBH suppresses CCA cell migration and invasion and decreases expression of proteins involved in cancer metastasis. RBH is a potential food supplement for cancer prevention.

Objective Cholangiocarcinoma (CCA) is a malignant tumor with aggressive metastatic property resulted from dysregulation of metastasis-regulated signaling pathways. The aim of this study was to investigate the effect of cucurbitacin B on metastatic behavior of CCA cells through modulation of focal adhesion kinase (FAK) protein. Methods KKU-452 cells were treated with a specific FAK inhibitor, FAK inhibitor-14, or cucurbitacin B at various concentrations for 24 h. Cell viability was assessed by sulforhodamine B assay. The migratory and invasive abilities of the cells were investigated using wound healing and transwell invasion assays, respectively. The fibronectin-coated plate was used for adhesion assay. The effects of the test compounds on FAK activation and the expression of metastasis-associated proteins were determined by Western blot analysis. The amount of MMP-9 was evaluated using a commercial ELISA Kit. Results FAK inhibitor-14 and cucurbitacin B at concentrations which minimally affected KKU-452 cell viability could suppress FAK activation, evidently by decreased level of phospho-FAK protein after exposure to the compound. At these conditions, cucurbitacin B suppressed metastatic behavior including migration, invasion and adhesion abilities of CCA cells similar to FAK inhibitor-14. Further molecular studies demonstrated that FAK inhibitor-14 and cucurbitacin B downregulated the expression of metastasis-associated proteins including MMP-9, ICAM-1 and VEGF. Consequently, exposure to cucurbitacin B inhibited the production of MMP-9 enzyme in CCA cells similar to FAK inhibitor-14 treatment. Conclusion FAK participated in regulation of metastatic behavior of KKU-452 CCA cells. Cucurbitacin B suppressed FAK activation in the cells which was associated with inhibition of metastasis essential steps and their related metastatic proteins. The compound may be developed as a novel therapeutic agent for CCA metastasis therapy. .

Phenformin (Phen), a potent activator of AMPK, is effective against some resistant cancers. This study evaluated the inhibition of proliferation, migration, invasion, and angiogenesis by Phen in aggressive cancer cells and investigated the underlying mechanism of the inhibition. Cholangiocarcinoma (CCA) KKU-156 and KKU-452 cells were used in this study. The results showed that Phen suppressed cell proliferation and induced apoptosis in both cells. Phen suppressed migration and invasion of cancer cells in wound healing and transwell chamber assays, respectively. The effects were associated with depletions of glutathione (GSH) and decreased glutathione redox ratio which represents cellular redox state. The redox stress was linked with the loss of mitochondrial transmembrane potential, as evaluated by JC-1 assay. The effect of Phen on angiogenesis was performed using HUVEC cultured cells. Phen alone did not affect tube formation of HUVEC cells. However, conditioned media from CCA cell cultures treated with Phen suppressed the tube-like structure formation. The antitumor effect of Phen was associated with AMPK activation and suppression of mTOR phosphorylation, HIF-1A, and VEGF protein expression. In conclusion, Phen inhibits cell proliferation, migration, invasion, and angiogenesis probably through AMPK-mTOR and HIF-1A-VEGF pathways. Phen may be repurposed as chemoprevention of cancer.

Objective: To evaluate the immunomodulatory effects of rice bran hydrolysates on cultured immune cells and their underlying mechanism. Methods: Rice bran hydrolysates were prepared from pigmented rice (Oryza sativa L.) by hydrothermolysis and protease digestion. Rice bran hydrolysates were assayed for phenolic content and antioxidant activity. Cell proliferation of Jurkat, THP-1 and peripheral blood mononuclear cells (PBMC) was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Chemotaxis was evaluated by transwell chamber methods. Immunoadherence of THP-1 was performed on cultured human umbilical vein endothelial cells (HUVEC). Cytokine released from PBMC was measured by ELISA assay kits. Lymphocyte-mediated cytotoxicity was carried out on KKU-452 cells. Proteins associated with immunomodulation were analyzed by Western immunoblotting assay. Results: Rice bran hydrolysates were rich in phenolic compounds, such as ferulic acid, catechin, quercetin, and quercetin glycosides. Rice bran hydrolysates suppressed phytohemagglutinin (PHA)- stimulated proliferation of PBMC and Jurkat cells, chemotaxis of Jurkat and THP-1 cells, and immunoadherence of THP-1 on HUVEC cultured cells. The cellular mechanism of rice bran hydrolysates involved the activation of AMPK as well as suppression of mTOR, NF-κB and VCAM-1. Rice bran hydrolysates potentiated PBMC on the PHA-stimulated release of IL-2, TNF-α, and IL-4, and enhanced PHA-induced non-MHC-restricted cytotoxicity on KKU-452 cancer cells. Conclusions: The immunomodulatory effect of phytochemicals derived from rice bran hydrolysates suggests its therapeutic potential for further investigation.

Pamidronate has been hypothesized to effectively inhibit cancer cell growth and metastasis in bone tissue. Furthermore, pamidronate (Pami) exerts various direct effects against several cancer cell types, including growth and migration. The present study aimed to determine the underlying mechanism of Pami's effect on the proliferation and migration of cholangiocarcinoma (CCA) cells. KKU-100 cells were used to determine the effects of Pami on cell death and migration. The following were assessed: Sulforhodamine B, colony formation, apoptosis via flow cytometry, reactive oxygen species (ROS) production and caspase-3 activity. In addition, the effects of the test compound on the mevalonate (MVA) signaling pathway were determined via western blotting and reverse transcription-quantitative PCR. Cell migration was observed via wound healing, Matrigel and gelatin zymography. The results indicated that Pami induced CCA cell death and inhibited colony formation in a dose-dependent manner, with IC50 values of 444.67±44.05 µM at 24 h and 147.33±17.01 µM at 48 h. Furthermore, Pami treatment suppressed colony formation at a lower concentration than growth inhibition with IC50 values of 5.36±0.31 µM. The mechanism of growth inhibition was determined to potentially be associated with increased ROS generation and stimulated caspase-3 enzyme activity, leading to the induction of apoptosis. Furthermore, Pami treatment interfered with the MVA signaling pathway by reducing Rac1 protein levels and modulating the gene and protein expression of RhoA. Furthermore, Pami suppressed CCA cell migration by decreasing matrix metalloproteinase (MMP)2 and MMP9 levels. Additionally, Pami treatment activated CCA cell death and inhibited CCA migration at low concentrations. Pami significantly decreased the protein expression levels of Rac1 in the MVA signaling pathway and may therefore be beneficial for developing a novel chemotherapeutic method for CCA.

Four new dimeric styrenes, 1-4, were isolated from an EtOAc crude extract of the seeds of Atalantia monophylla. The biosynthetic pathway of 1 is proposed to involve a [2 + 2] cycloaddition, while 2-4 may be generated via a polar mechanism with a carbocation as the key intermediate. The structures of 1-4 were defined from spectroscopic analysis; experimental and calculated ECD spectra were used to characterize their absolute configurations. When tested against two different cancer cell lines, 1-4 were not determined to be cytotoxic (IC50 > 10 μM).

Aims Mdivi-1, a selective Drp-1 inhibitor, impedes mitochondrial dynamics and suppresses cancer proliferation and progression. Cholangiocarcinoma (CCA) is a very aggressive malignancy which is refractory to chemotherapy. The study investigated the mechanism of the chemosensitizing effect of mdivi-1 in cholangiocarcinoma. Main methods CCA cells and HEK293 T cells were employed in the study. Cell viability and induction of apoptotic cell death were determined by the MTT and acridine orange-ethidium bromide methods. Cellular glutathione content and reactive oxygen species (ROS) formation were assessed using thiol green and 2′,7′-dichlorofluorescin diacetate fluorescent probes, respectively. Mitochondrial transmembrane potential and autophagy were detected by JC-1 dye and autophagy assay. Cell cycle progression was analyzed by flow cytometry. Cell migration was measured using the wound healing assay. Proteins involved in cell proliferation and cell cycle were analyzed by western immunoblotting. Key findings Mdivi-1 enhanced cisplatin-induced cytotoxicity in CCA cells but not in HEK293 T cells. Mdivi-1 enhanced cisplatin induced glutathione redox stress, ROS formation, and loss of mitochondrial transmembrane potential. Moreover, mdivi-1 also inhibited autophagic flux and suppressed CCA cell migration. Significance Mdivi-1 sensitized CCA cells to cytotoxicity of cisplatin in association with increases of oxidative stress and autophagosomes, and induced cell death via the mitochondrial pathway. Disruption of mitochondrial dynamics may be a novel strategy to improve the efficacy of chemotherapy to treat CCA.

Chemotherapy is a palliative treatment for unresectable patients with cholangiocarcinoma (CCA). However, drug resistance is a major cause of the failure of this treatment. Derrischalcone (DC), a novel chalcone isolated from Derris indica fruit, has been shown pharmacologically active; though, the effect of DC on CCA is unknown. The present study investigated the cytotoxic, antiproliferative, anti-migration, and anti-invasion effects and underlying mechanisms of DC on CCA KKU-M156 and KKU-100 cells. Cytotoxicity and apoptosis were evaluated by acridine orange and ethidium bromide fluorescent staining. Reactive oxygen species (ROS) was measured by dihydroethidium assay. Cell proliferation and reproductive cell death were assessed by sulforhodamine B staining and colony-forming assay. Migration and invasion were determined by wound healing and transwell chamber assays. Protein expressions associated with cell death, proliferation, migration, and invasion were analyzed by western immunoblotting. We found that DC induced cytotoxicity and apoptosis in association with ROS formation and oxidative stress. Treatment with N-acetylcysteine suppressed ROS formation and attenuated DC-induced cytotoxic and apoptotic effects. DC increased the expression of p53, p21, Bax, and cytochrome c proteins in association with cell death. DC-induced antiproliferation, colony formation, anti-migration, and anti-invasion were associated with the suppression of Akt/mTOR/cyclin D1 and FAK signaling pathways. These findings suggest that the multi-targeting strategies with DC may be a novel treatment for cancer therapy.

Lead (Pb) and cadmium (Cd) are environmental pollutants and nonessential elements in the body. Both metals induce the development of hypertension which is associated with oxidative stress. Curcumin (CUR) is a polyphenolic compound with strong antioxidant activity. The present study evaluated the effect of CUR on oxidative stress, alteration of vascular responsiveness and hypertension induced by exposure to either Pb, Cd or the combination of Pb and Cd. Male Sprague-Dawley rats were exposed to low level of lead acetate (100 mg/L) and/or cadmium chloride (10 mg/L) in the drinking water for 16 weeks. The control animals received deionized water as drinking water. CUR (100 mg/kg) or propylene glycol as vehicle was intragastrically administered once daily for the last 4 weeks. Exposure to Pb, Cd or the combination induced increases in blood pressure and peripheral vascular resistance, and decreased the blood pressure response to intravenous infusion to acetylcholine. Supplementation with CUR significantly reduced blood pressure, alleviated oxidative stress, and increased plasma nitrate/nitrite and glutathione in the blood. The effects of CUR were associated with the improvement of vascular responsiveness, upregulation of the endothelial nitric oxide synthase and downregulation of the NADPH oxidase expression. Furthermore, CUR reduced the metal levels in blood, aorta, liver and kidney. Altogether, exposure to the combination of Pb and Cd aggravated hypertension and oxidative stress, and CUR effectively ameliorated these adverse events in metal exposed animals. Data indicate that CUR may be useful as a dietary supplement for protection against the noxious effects of the heavy metals.

Inadequate responses to traditional chemotherapeutic agents in cholangiocarcinoma (CCA) emphasize a requirement for new effective compounds for the treatment of this malignancy. This study aimed to investigate the antiproliferative property of cucurbitacin B on KKU-100 CCA cells. The determination of underlying molecular mechanisms was also carried out. The results revealed that cucurbitacin B suppressed growth and replicative ability to form colonies of CCA cells, suggesting the antiproliferative effect of this compound against the cells. Flow cytometry analysis demonstrated that the interfering effect of cucurbitacin B on the CCA cell cycle at the G2/M phase was accountable for its antiproliferation property. Accompanied with cell cycle disruption, cucurbitacin B altered the expression of proteins involved in the G2/M phase transition including downregulation of cyclin A, cyclin D1, and cdc25A, and upregulation of p21. Additional molecular studies demonstrated that cucurbitacin B suppressed the activation of focal adhesion kinase (FAK) which consequently resulted in inhibition of its kinase-dependent and kinase-independent downstream targets contributing to the regulation of cell proliferation including PI3K/PDK1/AKT and p53 proteins. In this study, the transient knockdown of FAK using siRNA was employed to ascertain the role of FAK in CCA cell proliferation. Finally, the effect of cucurbitacin B on upstream receptor tyrosine kinases regulating FAK activation was elucidated. The results showed that the inhibitory effect of cucurbitacin B on FAK activation in CCA cells is mediated via interference of EGFR and HER2 expression. Collectively, cucurbitacin B might be a promising drug for CCA treatment by targeting FAK protein.

Rice bran hydrolysates contain highly nutritional proteins and beneficial phytochemicals. Sang-Yod rice bran hydrolysates (SRH) extracted from red pigmented rice is a rich source of nutrients and phenolic compounds. The present study evaluated the antihypertensive effect of SRH and its safety in Sprague-Dawley rats. Hypertension was induced in male rats by administration of L-NAME (50 mg/kg/day) in drinking water for three weeks, and the antihypertensive effect of SRH was evaluated. Treatment of SRH (250 or 500 mg/kg) significantly reduced arterial blood pressure and improved hemodynamic parameters. The antihypertensive effect was associated with decreased oxidative stress, suppressed p47phox NADPH oxidase expression, increased nitric oxide bioavailability and decreased angiotensin II level and ACE activity. The SRH was shown to be safe after feeding male and female rats with a rodent diet containing 1.5% SRH for 90 days. Overall, these findings suggest that SRH is safe and may help to prevent hypertension.

Twelve lignan derivatives were synthesized from deoxypodophyllotoxin isolated from Hernandia nymphaeifolia. Cytotoxicity evaluation against cholangiocarcinoma, KKU-100, and HepG2 cell lines showed that compounds 3, 9, 10, and 13 exhibited stronger cytotoxicity than the starting material, 1, with IC50 ranging from 0.42 to 2.01 μM. Compound 10 displayed interesting activity by showing IC50 values of 0.75 and 0.46 μM against KKU-100 and HepG2 cell lines, respectively. From these observation, 10 seems to be useful as a lead compound for the development of anticancer agents.

Aims Transcription factor Nrf2, which regulates the expression of cytoprotective and antioxidant enzymes, contributes to proliferation and resistance to chemotherapy in cancer. The inhibition of Nrf2 can sensitize cholangiocarcinoma (CCA) cells to the cytotoxicity of several chemotherapeutic agents. In this study, we investigated the mechanism of this chemosensitizing effect. Main methods KKU-100 cells were used in the study. Nrf2 expression was knocked down by siRNA and expression was validated by reverse transcription and polymerase chain reaction. Cytotoxicity was assessed by sulforhodamine B method. Intracellular reactive oxygen species (ROS) was examined by fluorescent dye, dichlorofluorescin diacetate method and mitochondrial transmembrane potential was assessed by JC1 dye assay. Key findings Cytotoxicity of cisplatin (Cis) in KKU-100 cells was enhanced by knockdown of Nrf2 expression. The enhanced cytotoxic effect was abolished by treatment with N-acetylcysteine, TEMPOL and MnTBAP. Cells with Nrf2 knockdown or Cis treatment increased production of ROS, and ROS was markedly enhanced by a combination of Nrf2 knockdown and Cis. The increased ROS formation was associated with a decrease in mitochondrial transmembrane potential (Δψm), where this decrease was prevented by antioxidant compounds. The loss of Δψm and cell death were prevented by cyclosporine, an inhibitor of mitochondrial permeability transition pore (MPTP). Luteolin inhibited Nrf2 and markedly enhanced cytotoxicity in combination with Cis. Significance Inhibition of Nrf2 is a feasible strategy in enhancing antitumor activity of chemotherapeutic agents and improving efficacy of chemotherapy in CCA.

Carthamus tinctorius L. (CT) is widely used in Asian countries as a beverage and in folk medicine. The effects of CT extract on hemodynamics, vascular remodeling, the renin-angiotensin system (RAS) and oxidative stress in the two-kidney, one clip (2K-1C) hypertensive rat model were investigated. Renovascular hypertension was induced in male Sprague-Dawley rats and were treated with CT extract (500mg/kg/day) or captopril (5mg/kg/day) or vehicle for four weeks. CT extract or captopril reduced blood pressure, hindlimb vascular resistance, and increased hindlimb blood flow in 2K-1C hypertensive rats (p

Hesperidin, a flavonoid derived from citrus fruits, possesses several beneficial effects including anti-oxidation and anti-inflammation. The aim of this study was to investigate the effects of hesperidin on the renin-angiotensin system (RAS) cascade that mediated oxidative stress and sympathoexcitation in two-kidney, one-clipped (2K-1C) hypertensive rats. 2K-1C hypertension was induced in male Sprague-Dawley rats. Hypertensive rats were treated with hesperidin at 20[Formula: see text]mg/kg or 40[Formula: see text]mg/kg or losartan at 10[Formula: see text]mg/kg beginning at three weeks after surgery and then continued for four weeks ([Formula: see text]/group). Hesperidin reduced blood pressure in a dose-dependent manner in hypertensive rats compared to untreated rats ([Formula: see text]). Increased plasma angiotensin converting enzyme (ACE) activity and angiotensin II levels, as well as, upregulated AT1 receptor protein expression in aortic tissues were attenuated in hypertensive rats treated with hesperidin. Hesperidin suppressed oxidative stress markers and NADPH oxidase over-expression, and restored plasma nitric oxide metabolites in 2K-1C rats. This was associated with improvement of the vascular response to acetylcholine in isolated mesenteric vascular beds and aortic rings from 2K-1C rats treated with hesperidin ([Formula: see text]). Enhancement of nerve-mediated vasoconstriction related to high plasma noradrenaline in the 2K-1C group was alleviated by hesperidin treatment ([Formula: see text]). Furthermore, losartan exhibited antihypertensive effects by suppressing the RAS cascade and oxidative stress and improved vascular dysfunction observed in 2K-1C rats ([Formula: see text]). Based on these results, it can be presumed that hesperidin is an antihypertensive agent. Its antihypertensive action might be associated with reducing RAS cascade-induced NOX2 over-expression and sympathoexcitation in 2K-1C hypertensive rats.

Asiatic acid, a triterpenoid compound derived from Centella asiatica, has been demonstrated to have antioxidant and anti-inflammatory effects. The present study evaluated the effects of asiatic acid on hemodynamic alterations, renin-angiotensin system (RAS), oxidative stress, and inflammation in 2K-1C hypertensive rats. Renovascular hypertension was induced in male Sprague-Dawley rats and treated with vehicle, asiatic acid (30 mg/kg/day), or captopril (5 mg/kg/day) for 4 weeks. We observed that 2K-1C hypertensive rats exhibited hemodynamic alterations such as high blood pressure, heart rate, hindlimb vascular resistance, and low hindlimb blood flow. Signs of RAS activation, such as increased plasma angiotensin II and serum angiotensin-converting enzyme activity, enhanced AT1R protein expression, and suppressed AT2R expression was observed in 2K-1C hypertensive rats. Overproduction of vascular superoxide, high levels of plasma MDA, low levels of plasma nitric oxide metabolites (NOx), and upregulation of gp91phox protein expression were observed in hypertensive rats. Furthermore, inflammation was observed in hypertensive rats, as evidenced by increased plasma TNF-α, NF-κB, and phospho-NF-κB protein expression. Asiatic acid or captopril alleviated hemodynamic alterations, RAS activation, oxidative stress, and inflammation in 2K-1C hypertensive rats. These findings indicate that asiatic acid is an antihypertensive agent that ameliorates hemodynamic alterations in 2K-1C hypertensive rats. This effect may involve one or both of the following mechanisms: the direct effect of asiatic acid on RAS activation, oxidative stress and inflammation, and/or asiatic acid acting as an ACE inhibitor agent to inhibit the Ang II-AT1R-NADPH oxidase-NF-κB pathway.

Nω-Nitro-l-arginine methyl ester (l-NAME)-induced hypertension and cardiovascular remodeling are associated with oxidative stress and inflammation. Garcinia mangostana Linn., has been reported to have antioxidant and anti-inflammatory properties. This study investigated whether G. mangostana pericarp extract (GME) could prevent l-NAME-induced hemodynamic alterations, cardiovascular remodeling, oxidative stress and inflammation in rats. Male Sprague-Dawley rats were given 40mg/kg/day of l-NAME in drinking water to induce hypertension, and were simultaneously treated with GME at a dose of 200mg/kg/day. Rats that received l-NAME for five weeks had high blood pressure, left ventricular hypertrophy and thickening of aortic wall. Vascular superoxide production, plasma malondialdehyde (MDA), and plasma tumor necrosis factor alpha (TNF-α) were significantly increased in l-NAME-hypertensive rats (p

Excessive iron can generate reactive oxygen species (ROS), leading to oxidative stress that is closely associated with cardiovascular dysfunction. Iron overload was induced in male ICR mice by injection of iron sucrose (10mg/kg/day) for eight weeks. Iron overload was evidenced by increased serum iron indices. The mice developed increased blood pressure, impaired vascular function and blunted response of the autonomic nervous system. These effects were accompanied by increased malondialdehyde levels in various tissues, increased nitric oxide metabolites in plasma and urine, and decreased blood glutathione. Tetrahydrocurcumin (THU, 50mg/kg/day), deferiprone (or L1, 50mg/kg/day) or both was orally administered throughout the period of iron sucrose injection. The treatments significantly alleviated the deleterious cardiovascular effects of iron overload, and were associated with modulation of nitric oxide levels. An imbalance between endothelial nitric oxide synthase (eNOS) and inducible NOS (iNOS) expression in response to iron overload was normalized by THU, L1 or the combination treatment. Moreover, the treatment decreased the upregulated expression levels of gp91phox, p47phox and HO-1. The combination of THU and L1 exerted a greater effect than THU or L1 monotherapy. These results suggest beneficial effects of THU and L1 on iron-induced oxidative stress, hypertension, and vascular dysfunction.

Oxidative stress caused as a result of iron overload is implicated in clinical manifestation of beta-thalassemia/haemoglobin E (β-Thal/HbE). In this study, we investigated the cellular adaptation against oxidative stress in β-Thal/HbE patients. Twenty-four paediatric β-Thal/HbE patients and 22 healthy controls were recruited in the study. Blood samples from patients exhibited iron overload, elevation of lipid peroxidation, and marked diminution in the reduced glutathione (GSH) level. However, expression of glutamate-cysteine ligase catalytic (GCLC) subunit, a key enzyme in GSH biosynthesis, was up-regulated when compared with that in controls. GCLC protein levels were correlated with serum iron. There was an enhanced binding activity of the oligonucleotide probe for Nrf2-driven antioxidant response element (ARE) to nuclear protein from blood mononuclear cells of thalassemia subjects. In conclusion, β-Thal/HbE patients exhibit elevated plasma levels of GCLC expression and Nrf2-ARE binding activity, which may account for their adaptive survival response to oxidative stress.

Aim of Study: Cholangiocarcinoma (CCA) is an aggressive cancer with considerable metastatic potential. Various cytokines secreted by tumor cells or cells in the tumor environment can promote the metastasis of CCA. The aim of the present study was to investigate the effect of myricetin on the inhibition of cytokine-induced migration and invasion and the associated cellular mechanisms in human CCA cells. Materials and Methods: CCA KKU-100 cells were treated with a pro-inflammatory cytokine mixture consisting of interleukin-6, interferon-γ, and tumor necrosis factor-α. The migratory and invasive ability of KKU-100 cells were determined using a wound-healing assay and transwell invasion assay. The effect of myricetin on cytokine-induced STAT3 activation in CCA cells was determined using Western blot analysis. The real-time polymerase chain reaction was performed to determine messenger RNA expression. Results: Myricetin significantly inhibited cytokine-induced migration and invasion of KKU-100 cells. Detailed molecular analyses revealed that myricetin suppressed the activation of the STAT3 pathway, evidently by a decrease of the active phospho-STAT3 protein expression after myricetin treatment. The cytokine-mediated upregulation of metastasis- and inflammatory-associated genes, which are downstream genes of STAT3 including the intercellular adhesion molecule-1, matrix metalloproteinase-9, inducible nitric oxide synthase, and cyclo-oxygenase 2 (COX-2), were also significantly abolished by myricetin treatment. Moreover, the anti-migratory and anti-invasive activities of a widely prescribed COX inhibitor, indomethacin, were also revealed. Conclusion: This finding reveals the anti-metastatic effect of myricetin against CCA cells which is mediated partly through suppression of the STAT3 pathway. This compound could be potentially useful as a therapeutic agent against CCA.

Objective Endothelial dysfunction associated with reduction in nitric oxide (NO) bioavailability plays an important role in development of hypertension. Consumption of a diet rich in antioxidants appears to lower the risk for hypertension. Virgin rice bran oil (VRBO) possesses antioxidant, anti-inflammatory, and hypocholesterolemic activities. However, to our knowledge, the antihypertensive effect of VRBO has not been investigated. The aim of this study was to examine the antihypertensive effect of VRBO in Nω-nitro- l -arginine methyl ester (L-NAME)-induced hypertensive rats and its underlying mechanisms. Methods Hypertension was induced in rats by administration of L-NAME, after which VRBO, lisinopril (Lis), or VRBO + Lis was administered. Studies were then conducted on the hemodynamics of vascular responses to vasoactive substances, plasma angiotensin-converting enzyme (ACE), plasma nitrate/nitrite, oxidative stress, and inflammatory markers. Results L-NAME administration induced hemodynamic changes including elevation of blood pressure, increased peripheral vascular resistance, and endothelial dysfunction. Reduction in plasma nitrate/nitrite, overproduction of vascular superoxide, and increases in plasma ACE, malondialdehyde, protein carbonyl, and plasma tumor necrosis factor-α were observed in L-NAME hypertensive rats. The changes were associated with a marked decrease in endothelial NO synthase expression, increased expression of gp91phoxand vascular cell adhesion molecule-1, and activation of nuclear factor-κB in aortic tissues. Administration of either VRBO or Lis significantly mitigated all of these deleterious effects. The combination of VRBO and Lis was more effective than either treatment alone. Conclusions The antihypertensive effect of VRBO may be mediated by restoration of hemodynamics, increased NO bioavailability, and alleviation of oxidative stress and inflammation. VRBO has an additive effect on antihypertensive medication.

Altered expression of a cytosolic flavoenzyme NAD(P)H:quinone oxidoreductase-1 (NQO1) has been seen in many human tumors. Its remarkable overexpression in cholangiocarcinoma (CCA; an aggressive malignancy of the biliary duct system) was associated with poor prognosis and short survival of the patients. Inhibition of NQO1 has been proposed as a potential strategy to improve the efficacy of anticancer drugs in various cancers including CCA. This study investigated novel NQO1 inhibitors and verified the mechanisms of their enzyme inhibition. Among the different chemical classes of natural NQO1 inhibitors are coumarins, flavonoids, and triterpenoids. Coumarins are a group of particularly potent NQO1 inhibitors. The mechanisms and kinetics of enzyme inhibition of coumarin, aesculetin, umbelliferone, and scopoletin using the cell lysates as a source of NQO1 enzyme best fit with an uncompetitive inhibition model. Among the NOQ1 inhibitors tested in KKU-100 CCA cells, scopoletin and umbelliferone had the strongest inhibitory effect on this enzyme, while aesculetin and coumarin barely affected intracellular NQO1. All coumarins were further tested for cytotoxicity and anti-migration activity. At modest cytotoxic doses, scopoletin and umbelliferone greatly inhibited the migration of KKU-100 cells, whereas coumarin and aesculetin barely reduced cell migration. The anti-migration effect of scopoletin was associated with decreased ratio of matrix metalloproteinase 9/tissue inhibitors of metalloproteinases 1 ( MMP9/ TIMP1) mRNA. These findings suggest that natural compounds with potent inhibitory effect on intracellular NQO1 have useful anti-migration effects on CCA cells. In order to prove that the potent NQO1 inhibitor, scopoletin, is clinically useful in the enhancement of CCA treatment, additional in vivo studies to elucidate the mechanism of these effects are needed.

Microphthalmia-associated transcription factor (MITF) is a key regulator of differentiation of melanocytes and retinal pigment epithelial cells, but it also has functions in non-pigment cells. MITF consists of multiple isoforms, including widely expressed MITF-A and MITF-H. In the present study, we explored the potential role played by the Hedgehog signaling on MITF expression in two common types of primary liver cancer, using human cholangiocarcinoma cell lines, the KKU-100 and HuCCT1, along with the HepG2 human hepatocellular carcinoma cell line. Importantly, cholangiocarcinoma is characterized by the activated Hedgehog signaling. Here we show that MITF-A mRNA is predominantly expressed in all three human liver cancer cell lines examined. Moreover, cyclopamine, an inhibitor of the Hedgehog signalling, increased the expression levels of MITF proteins in HuCCT1 and HepG2 cells, but not in KKU-100 cells, suggesting that MITF expression may be down-regulated in some liver cancer cases.

Cadmium (Cd) is a non-essential heavy metal with high toxicity potential. Humans are exposed to Cd present in diet, polluted air, and cigarette smoke. Cd exposure has been associated with increased risk of chronic diseases, including hypertension, atherosclerosis, diabetes, and nephropathy, all of which could be attributable to dysfunctional endothelial and smooth muscle cells. Cd toxicity is correlated with increased reactive oxygen formation and depletion of antioxidants, resulting in an oxidative stress. Chelation of Cd has proved useful in the removal of the Cd burden. However, several chelating agents cause side effects in clinical usage. Recent studies have shown that the antioxidant compounds curcumin and tetrahydrocurcumin can alleviate vascular dysfunction and high blood pressure caused by Cd toxicity. In chronic Cd exposure, these antioxidants protect vascular endothelium by increasing nitric oxide (NO•) bioavailability and improving vascular function. Antioxidant activity against Cd intoxication results directly and/or indirectly through free radical scavenging, metal chelation, enhanced expression of the antioxidant defense system, regulation of inflammatory enzymes, increase in NO• bioavailability, and reduction of gastrointestinal absorption and tissue Cd accumulation. This review summarizes current knowledge of Cd-induced oxidative stress and cardiovascular dysfunction and a possible protective effect conferred by the antioxidants curcumin and tetrahydrocurcumin.

Low efficacy and high resistance rate associated with existing chemotherapeutic drugs enforce a requirement for novel therapeutic strategies for extremely aggressive cholangiocarcinoma (CCA). In the present study, the apoptosis-inducing activity of cucurbitacin B, a compound derived from plants of Cucurbitaceae family, against KKU-100 CCA cells and the underlying mechanism mediating its effect were investigated. The results showed that cucurbitacin B significantly decreased CCA cells viability by induction of apoptosis. Increased apoptotic cell death following cucurbitacin B treatment was correlated with caspase-9 and caspase-3 activations, Bax upregulation, increased cytochrome c, apoptosis-inducing factor release, and decreased Bcl-2 and Bcl-XL levels, suggesting activation of the mitochondrial-mediated apoptosis pathway. Further molecular analyses revealed that cucurbitacin B inhibited focal adhesion kinase (FAK), which is an important regulator of the apoptosis process, and its downstream pathway, PI3K/Akt. Knockdown of FAK expression by small interfering RNA appeared to induce CCA cell apoptosis which was accompanied with elevated level of cytochrome c and cleaved caspase-9, and decreased level of Bcl-2, phospho-PI3K, and phospho-Akt. Taken together, cucurbitacin B induces an intrinsic mitochondrial apoptosis pathway in CCA cells partly through suppression of FAK-mediated oncogenic signaling. This compound should be considered as a candidate agent for CCA treatment.

Cymbopogon citratus (DC) Stapf., commonly known as lemongrass, possesses strong antioxidant and cardiotonic properties. Lemongrass water extract contains several polyphenolic compounds including gallic acid, isoquercetin, quercetin, rutin, catechin and tannic acid. Rutin, isoquercetin catechin and quercetin are the flavonoids most abundantly found in the extract. The extract significantly decreased total cholesterol, low-density lipoprotein and atherogenic index in rats after treatment (p < 0.05). Expression of genes and protein of sterol regulatory element binding protein-1c (SREBP1c) and HMG-CoA reductase (HMGR) was also lowered significantly in treated groups (p < 0.05). Moreover, serum antioxidant capacity increased in treated rats in comparison with untreated ones (p < 0.05) and was associated with decreased serum lipid peroxidation.

Phenethyl isothiocyanate (PEITC) is a potential cancer prevention agent that is found in cruciferous vegetables. Previous studies have shown that the effect of PEITC-induced cell death declines rapidly after administration. The metabolic fate of PEITC is modulated by glutathione S-transferases (GST). In this study, we investigated whether GST activity modulates PEITC-induced cytotoxicity on cholangiocarcinoma (CCA) cells. The sensitivity of KKU-M214 and KKU-100 cells to PEITC was associated with GST activity. Two GST inhibitors, ethacrynic acid (EA) and cibacron blue, potentiated the cytotoxic effect of PEITC in CCA cells. PEITC-induced glutathione (GSH) depletion and redox stress, whereas EA itself or in combination with PEITC did not alter GSH redox status. The enhanced cytotoxic effect of EA may be due to inhibition of GST activity. This idea was validated by using siRNA directed against GSTP1 mRNA in KKU-M214 cells, and GSTP1 and GSTT1 mRNA in KKU-100 cells. These GST isoforms were abundantly expressed in the cell lines. Knockdown of GSTs in CCA cell lines potentiated the cytotoxic effect of PEITC. The present study shows that the antitumor effect of PEITC was potentiated by the suppression of GST activity. The inhibition of GST could be a crucial strategy to potentiate chemotherapeutic effect of PEITC on CCA.

Summary A previous study demonstrated the antihypertensive effect of asiatic acid. The current study investigates the effect of asiatic acid on cardiovascular remodelling and possible mechanisms involved in Nω-nitro-L-arginine methyl ester hydrochloride (L-NAME)-induced hypertensive rats. Male Sprague–Dawley rats were treated with L-NAME (40 mg/kg per day) for 3 weeks in order to induce hypertension. Hypertensive rats were administered asiatic acid (20 mg/kg per day) or vehicle for a further 2 weeks. It was found that hypertensive rats showed high systolic blood pressure, left ventricular (LV) hypertrophy, increases in LV fibrosis, aortic wall thickness and aortic collagen deposition (P

In recent years, a number of studies have investigated complementary medical approaches to the treatment of hypertension using dietary supplements. Rice bran protein hydrolysates extracted from rice is a rich source of bioactive peptides. The present study aimed to investigate the vasorelaxation and antihypertensive effects of peptides-derived from rice bran protein hydrolysates (RBP) in a rat model of two kidney-one clip (2K-1C) renovascular hypertension. 2K-1C hypertension was induced in male Sprague-Dawley rats by placing a silver clip around the left renal artery, whereas sham-operated rats were served as controls. 2K-1C and sham-operated rats were intragastrically administered with RBP (50 mg kg−1 or 100 mg kg−1) or distilled water continuously for six weeks. We observed that RBP augmented endothelium-dependent vasorelaxation in all animals. Administration of RBP to 2K-1C rats significantly reduced blood pressure and decreased peripheral vascular resistance compared to the sham operated controls (p &lt, 0.05). Restoration of normal endothelial function and blood pressure was associated with reduced plasma angiotensin converting enzyme (ACE), decreased superoxide formation, reduced plasma malondialdehyde and increased plasma nitrate/nitrite (p &lt, 0.05). Up-regulation of eNOS protein and down-regulation of p47phox protein were found in 2K-1C hypertensive rats-treated with RBP. Our results suggest that RBP possesses antihypertensive properties which are mainly due to the inhibition of ACE, and its vasodilatory and antioxidant activity.

Three new limonoids, limonophyllines A-C (1, 4 and 5), along with two known limonoids (2 and 3) and 11 acridone alkaloids (6-16) were isolated from the stems of Atalantia monophylla. All isolates were evaluated against cholangiocarcinoma, KKU-M156, and HepG2 cancer cell lines. Compounds 12, 14 and 16 displayed cytotoxicity against KKU-M156 cell line with IC50 ranging from 3.39 to 4.1 μg/mL while cytotoxicity against HepG2 cell line with IC50 ranging from 1.43 to 8.4 μg/mL. The structures of all isolated compounds were established by spectroscopic methods including 1D and 2D NMR, IR and mass spectrometry.

Chemical investigation of the ethyl acetate extract from the fruits of Derris indica has led to the isolation of a new furanoflavonoid derivative, 4′-hydroxypinnatin (1), and five known compounds. Pinnatin (2) showed strong cytotoxicity against cholangiocarcinoma (KKU-100) and human hepatoma (HepG2) cell lines with IC50 values of 6.0 ± 2.7 and 9.0 ± 4.1 μg/ml, respectively, and showed maximal cell killing effect of about 88–90%. Flavone 5 exhibited the most cytotoxicity against KKU-100 but it showed moderate efficacy (Emax = 50.7%).

We investigated the effect of asiatic acid (AA) on hemodynamic status, vascular function, oxidative stress markers, endothelial nitric oxide synthase (eNOS), and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase subunit expression in Nω-nitro-l-arginine methyl ester hydrochloride (l-NAME)-induced hypertensive rats. Male Sprague–Dawley rats treated with l-NAME (40 mg/kg/day) in drinking water for 5 weeks showed significant increases in mean arterial pressure, heart rate, hindlimb vascular resistance, vascular dysfunction, superoxide anion (O2•−) production, and plasma malondialdehyde. Moreover, NO metabolite (NOx) levels were reduced, aortic eNOS expression was downregulated, and NADPH oxidase subunit p47phox was upregulated in hypertensive rats (p

Asiatic acid is a triterpenoid isolated from Centella asiatica. The present study aimed to investigate whether asiatic acid could lessen the metabolic, cardiovascular complications in rats with metabolic syndrome (MS) induced by a high-carbohydrate, high-fat (HCHF) diet. Male Sprague-Dawley rats were fed with HCHF diet with 15% fructose in drinking water for 12 weeks to induce MS. MS rats were treated with asiatic acid (10 or 20 mg/kg/day) or vehicle for a further three weeks. MS rats had an impairment of oral glucose tolerance, increases in fasting blood glucose, serum insulin, total cholesterol, triglycerides, mean arterial blood pressure, heart rate, and hindlimb vascular resistance; these were related to the augmentation of vascular superoxide anion production, plasma malondialdehyde and tumor necrosis factor-alpha (TNF-α) levels (p < 0.05). Plasma nitrate and nitrite (NOx) were markedly high with upregulation of inducible nitric oxide synthase (iNOS) expression, but dowregulation of endothelial nitric oxide synthase (eNOS) expression (p < 0.05). Asiatic acid significantly improved insulin sensitivity, lipid profiles, hemodynamic parameters, oxidative stress markers, plasma TNF-α, NOx, and recovered abnormality of eNOS/iNOS expressions in MS rats (p < 0.05). In conclusion, asiatic acid improved metabolic, hemodynamic abnormalities in MS rats that could be associated with its antioxidant, anti-inflammatory effects and recovering regulation of eNOS/iNOS expression.

Phenethyl isothiocyanate (PEITC) is a natural isothiocyanate with anticancer activity against many drug-resistant cancer cells. A body of evidence suggests that PEITC enhances oxidative stress leading to cancer cell death. Cholangiocarcinoma (CCA) is an aggressive bile duct cancer with resistance to chemotherapeutic drugs. PEITC rapidly kills KKU-100 CCA cells with concurrent induction of cellular glutathione depletion, superoxide formation, and loss of mitochondrial transmembrane potential. The loss was associated with increased Bax and decreased Bcl-xl proteins followed by the release of cytochrome c and the activation of caspase-9 and -3. Although TEMPOL could prevent superoxide formation, it did not prevent the disruption of glutathione (GSH) redox, mitochondrial dysfunction, and cell death. On the other hand, N-acetylcysteine could prevent the events and cell death. It was concluded that disruption of GSH redox but not superoxide formation may be an initial step leading to mitochondrial injury. PEITC could be a promising chemopreventive agent for CCA.

Diabetic nephropathy (DN) is an important microvascular complication of uncontrolled diabetes. The features of DN include albuminuria, extracellular matrix alterations, and progressive renal insufficiency. Rice bran protein hydrolysates (RBPs) have been reported to have antihyperglycemic, lipid-lowering, and anti-inflammatory effects in diabetic rats. Our study was to investigate the renoprotective effects of RBP in diabetic animals and mesangial cultured cells. Eight-week-old male db/m and db/db mice were orally treated with tap water or RBP (100 or 500 mg/kg/day) for 8 weeks. At the end of the experiment, diabetic nephropathy in kidney tissues was investigated for histological, ultrastructural, and clinical chemistry changes, and biomarkers of angiogenesis, fibrosis, inflammation, and antioxidant in kidney were analyzed by Western blotting. Protection against proangiogenic proteins and induction of cytoprotection by RBP in cultured mesangial cells was evaluated. RBP treatment improved insulin sensitivity, decreased elevated fasting serum glucose levels, and improved serum lipid levels and urinary albumin/creatinine ratios in diabetic mice. RBP ameliorated the decreases in podocyte slit pore numbers, thickening of glomerular basement membranes, and mesangial matrix expansion and suppressed elevation of MCP-1, ICAM-1, HIF-1α, VEGF, TGF-β, p-Smad2/3, and type IV collagen expression. Moreover, RBP restored suppressed antioxidant Nrf2 and HO-1 expression. In cultured mesangial cells, RBP inhibited high glucose-induced angiogenic protein expression and induced the expression of Nrf2 and HO-1. RBP attenuates the progression of diabetic nephropathy and restored renal function by suppressing the expression of proangiogenic and profibrotic proteins, inhibiting proinflammatory mediators, and restoring the antioxidant and cytoprotective system.

Rice bran protein hydrolysates (RBPH) contain highly nutritional proteins and antioxidant compounds which show benefits against metabolic syndrome (MetS). Increased arterial stiffness and the components of MetS have been shown to be associated with an increased risk of cardiovascular disease. This study aimed to investigate whether RBPH could alleviate the metabolic disorders, arterial stiffening, vascular remodeling, and oxidative stress in rats fed a high-carbohydrate and high-fat (HCHF) diet. Male Sprague–Dawley rats were fed either a standard chow and tap water or a HCHF diet and 15 % fructose solution for 16 weeks. HCHF rats were treated orally with RBPH (250 or 500 mg/kg/day) for the final 6 weeks of the experimental period. Rats fed with HCHF diet had hyperglycemia, insulin resistance, dyslipidemia, hypertension, increased aortic pulse wave velocity, aortic wall hypertrophy and vascular remodeling with increased MMP-2 and MMP-9 expression. RBPH supplementation significantly alleviated these alterations (P

This study examined the effect of Carthamus tinctorius (CT) extract plus captopril treatment on blood pressure, vascular function, nitric oxide (NO) bioavailability, oxidative stress and renin-angiotensin system (RAS) in N(ω)-Nitro-l-arginine methyl ester (l-NAME)-induced hypertension. Rats were treated with l-NAME (40 mg/kg/day) for five weeks and given CT extract (75 or 150 or 300 or 500 mg/kg/day): captopril (5 mg/kg/day) or CT extract (300 mg/kg/day) plus captopril (5 mg/kg/day) for two consecutive weeks. CT extract reduced blood pressure dose-dependently, and the most effective dose was 300 mg/kg/day. l-NAME-induced hypertensive rats showed abnormalities including high blood pressure, high vascular resistance, impairment of acetylcholine-induced vasorelaxation in isolated aortic rings and mesenteric vascular beds, increased vascular superoxide production and plasma malondialdehyde levels, downregulation of eNOS, low level of plasma nitric oxide metabolites, upregulation of angiotensin II type 1 receptor and increased plasma angiotensin II. These abnormalities were alleviated by treatment with either CT extract or captopril. Combination treatment of CT extract and captopril normalized all the abnormalities found in hypertensive rats except endothelial dysfunction. These data indicate that there are synergistic antihypertensive effects of CT extract and captopril. These effects are likely mediated by their anti-oxidative properties and their inhibition of RAS.

Mulberry (Morus alba L.) leaf tea is promoted for its health benefits and the control of diabetes in Asian nations. The blood glucose lowering activity of mulberry leaf extract (MA) has been proven; however, the molecular basis underlying this effect remains unclear. The aim of the present work is to elucidate its mechanism of the antihyperglycemic action, by examining the effect of MA on glucose uptake and the translocation of glucose transporter 4 protein (GLUT4) to the plasma membrane of adipocytes isolated from diabetic rats. The incubation of adipocytes with 5–45 μg/ml MA resulted in 31–54% increase of glucose uptake in a dose-dependent manner. This glucose uptake enhancing effect was inhibited by the phosphoinositol 3-kinase (PI3-K) inhibitor, wortmannin (100 nM). The GLUT4 protein on the plasma membrane fraction of adipocytes was markedly increased after treatment with 15 μg/ml MA extract. Interestingly, gallic acid, one of the phenolic compounds found in MA extract, increased glucose uptake and enhanced the translocation of GLUT4 at concentrations comparable to the amount of gallic acid in the effective concentration ranges of MA. Thus, it is likely that gallic acid contributes, at least in part, to its antihyperglycemic activity. The present results suggest that the antihyperglycemic action of MA is mediated by increasing glucose uptake via the activation of PI3-K signaling pathway and translocation of GLUT4 to the plasma membrane. These findings are the first molecular evidence supporting the mulberry tea as herbal medicine for diabetic patients.

Objectives To investigate the apoptosis-inducing effect and underlying mechanisms of luteolin in cholangiocarcinoma (CCA) cells. Methods Cell viability was determined by sulphorhodamine B. Apoptosis was detected using acridine orange/ethidium bromide dye staining and annexin V/PI staining followed by flow cytometry. The effect of luteolin on the oxidative status of CCA cells was evaluated by measuring intracellular reactive oxygen species (ROS) levels using the dihydroethidium method and quantifying glutathione levels. The mitochondria transmembrane potential (ΔΨm) was examined through JC-1 staining. The protein levels were determined by Western blot. Caspase activity was determined using specific fluorogenic substrates. Key findings Luteolin decreased KKU-100 CCA cells’ viability by induction of apoptosis. Luteolin treatment increased ROS production and decreased glutathione levels. These changes were associated with the decrease of Nrf2, γ-glutamylcysteine ligase and heme oxygenase-1 proteins. Moreover, luteolin induced mitochondrial depolarization, which was accompanied by the release of cytochrome c and a decrease of Bcl-2 and Bcl-XL proteins. Pretreatment with antioxidants, 4-hydroxy-TEMPO and N-acetyl-L-cysteine significantly prevented luteolin-induced CCA cell death and loss of ΔΨm. In addition, luteolin induced the activation of caspase-9 and caspase-3. Conclusions Luteolin exerts its pro-apoptotic action partly through generating intracellular ROS that then contributes to the activation of mitochondria-mediated apoptotic cell death.

A new 7-oxygenated coumarin, 7-demethylmurralonginol isovalerate (1), and a new natural product, murralonginol (2), together with seven known 7-oxygenated coumarins, murralonginol isovalerate (3), murralongin (4), micromelin (5), scopoletin (6), microminutin (7), murrangatin (8), and minumicrolin (9), were isolated from the fruits of Micromelum minutum. The structures of these compounds were established on the basis of their 1D and 2D NMR spectroscopic data. Among these isolates, compounds 2 and 4 - 9 exhibited cytotoxicity against cholangiocarcinoma cell line, KKU-100.

Several vegetables have been shown to possess cytoprotective and antioxidant effects with various mechanisms of action. The aim of this study was to determine the antioxidant effects and mechanism underlying of Syzygium gratum, a dietary and herbal plant commonly found in the Southeast Asia. Additionally, its effects on the induction of endogenous antioxidant defensive system were also investigated. Results showed that the leaf extract possessed an exceptionally strong antioxidant and intracellular oxygen radical scavenging activity in both aqueous and ethanolic extracts. The plant aqueous extract was further studied in C57BL/6J mice to evaluate its effects in vivo. The extract was well tolerated by the animals throughout the 30 days of study. The cytoprotective enzyme, heme oxygenase (HO-1) activity was significantly increased in the high dose-treated animals (1 g/kg/day). Consistent with the enzymatic activity, the expression of HO-1 mRNA tended to increase in those mice. There was no significant increase in hepatic γ-glutamylcysteine ligase (γ-GCL) activity, glutathione levels and GCL mRNA expression. Taken together, this study provides evidence that S. gratum exhibits potent direct antioxidant properties and can induce cytoprotective enzyme in vivo. Consumption of S. gratum may provide a health benefit against oxidative stress and other related disorders.

Hesperidin is a major flavonoid isolated from citrus fruits that exhibits several biological activities. This study aims to evaluate the effect of hesperidin on cardiovascular remodeling induced by n-nitro l-arginine methyl ester (l-NAME) in rats. Male Sprague-Dawley rats were treated with l-NAME (40 mg/kg), l-NAME plus hesperidin (15 mg/kg), hesperidin (30 mg/kg), or captopril (2.5 mg/kg) for five weeks (n = 8/group). Hesperidin or captopril significantly prevented the development of hypertension in l-NAME rats. l-NAME-induced cardiac remodeling, i.e., increases in wall thickness, cross-sectional area (CSA), and fibrosis in the left ventricular and vascular remodeling, i.e., increases in wall thickness, CSA, vascular smooth muscle cells, and collagen deposition in the aorta were attenuated by hesperidin or captopril. These were associated with reduced oxidative stress markers, tumor necrosis factor-alpha (TNF-&alpha, ), transforming growth factor-beta 1 (TGF-&beta, 1), and enhancing plasma nitric oxide metabolite (NOx) in l-NAME treated groups. Furthermore, up-regulation of tumor necrosis factor receptor type 1 (TNF-R1) and TGF- &beta, 1 protein expression and the overexpression of matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9) was suppressed in l-NAME rats treated with hesperidin or captopril. These data suggested that hesperidin had cardioprotective effects in l-NAME hypertensive rats. The possible mechanism may involve antioxidant and anti-inflammatory effects.

Cadmium (Cd) is one of the most important environmental pollutants that cause a number of adverse health effects in humans and animals. Recent studies have shown that Cd-induced oxidative damage within the vascular tissues results in vascular dysfunction. The current study was aimed to investigate whether ascorbic acid could protect against Cd-induced vascular dysfunction in mice. Male ICR mice were received CdCl(2) (100 mg/l) via drinking water for 8 weeks alone or received ascorbic acid supplementation at doses of 50 and 100 mg/kg/day for every other day. Results showed that Cd administration increased arterial blood pressure and blunted the vascular responses to vasoactive agents. These alterations were related to increased superoxide production in thoracic aorta, increased urinary nitrate/nitrite, increased plasma protein carbonyl, elevated malondialdehyde (MDA) concentrations in plasma and tissues, decreased blood glutathione (GSH), and increased Cd contents in blood and tissues. Ascorbic acid dose-dependently normalized the blood pressure, improved vascular reactivities to acetylcholine (ACh), phenylephrine (Phe) and sodium nitroprusside (SNP). These improvements were associated with significant suppression of oxidant formation, prevention of GSH depletion, and partial reduction of Cd contents in blood and tissues. The findings in this study provide the first evidence in pharmacological effects of ascorbic acid on alleviation of oxidative damage and improvement of vascular function in a mouse model of Cd-induced hypertension and vascular dysfunction. Moreover, our study suggests that dietary supplementation of ascorbic acid may provide beneficial effects by reversing the oxidative stress and vascular dysfunction in Cd-induced toxicity.

Cadmium (Cd) is a heavy metal which causes concern as an environmental toxicant. Therapy with chelating agents is considered to be the rational treatment against metal poisoning. This study was designed to evaluate whether meso-2,3-dimercaptosuccinic acid (DMSA) could alleviate oxidative stress and vascular dysfunction in mice with subchronic exposure to Cd. Male ICR mice received CdCl2 (100 mg/L) via drinking water for 8 weeks. After Cd exposure, DMSA at a dose of 25 mg/kg or 50 mg/kg was intragastrically administered once daily for 5 consecutive days at the end of Cd treatment. It was found that Cd-induced hypertension and markedly blunted vascular responses to vasoactive agents, including acetylcholine, phenylephrine and sodium nitroprusside. Treatment with DMSA significantly restored blood pressure and improved vascular responsiveness when compared with Cd-treated controls. Moreover, DMSA protected against Cd-induced severe oxidative stress by normalization of the redox ratios of glutathione to glutathione disulfide and suppression of plasma malondialdehyde, plasma protein carbonyl, urinary nitrate/nitrite, and superoxide production from thoracic aorta. DMSA partially reduced Cd contents in the blood, heart, liver and kidneys. In conclusion, our present study provides the first evidence of the therapeutic efficacy of DMSA against oxidative stress and vascular dysfunction in Cd-intoxicated mice.

Purpose : To investigate the antitumor effect of candidone extracted from Derris indica , against human cholangiocarcinoma (CCA) cells. Methods : Candidone was purified from the hexane extract of Derris indica fruit. CCA cell lines, KKUM156 and KKU-M213, were treated with candidone. Sulforhodamine B (SRB) assay and acridine orange/ethidium bromide (AO/EB) staining were used to investigate the effects of candidone on cell proliferation and induction of apoptosis, respectively. The effect on cell migration was assessed by wound healing assay. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was employed to assess the effects of candidone on the expression of genes that regulate proliferation and apoptosis. Results : Candidone exerted strong anticancer effects on CCA cells. The agent suppressed CCA cell proliferation and induced apoptotic cell death. RT-qPCR assay revealed that candidone significantly increased the expression of anti-proliferative and pro-apoptotic genes, including p21 and Bax, and decreased the expression of anti-apoptotic genes, including Bcl-2 and survivin. Moreover, candidone inhibited the migration of CCA cells induced by IGF-1. Conclusion : Candidone exhibits potent antitumor effect on CCA cells. These findings suggest that candidone is potentially suitable for the management of CCA and, therefore, warrants further investigation. Keywords : Candidone, Derris indica, Cholangiocarcinoma, Cytotoxicity, Apoptosis