Phikud Navakot extract attenuates lipopolysaccharide-induced inflammatory responses through inhibition of ERK1/2 phosphorylation in a coculture system of microglia and neuronal cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Phikud Navakot (PN), a mixture of nine herbal plants, is an ancient Thai traditional medicine used for relieving circulatory disorders and dizziness. PN has also shown anti-inflammatory effects in rats with acute myocardial infarction. Moreover, phytochemical-inhibiting neuroinflammation, including gallic acid, vanillic acid, ferulic acid, and rutin were detected in PN extract; however, the anti-neuroinflammatory activity of PN extract and its components in a coculture system of microglia and neuronal cells is limited. OBJECTIVE: To investigate the anti-neuroinflammatory activities of PN on lipopolysaccharide (LPS)-induced inflammation in a coculture system of microglia and neuronal cells. METHODS: ELISA and qRT-PCR were used to assess cytokine expression. The phosphorylation of mitogen-activated protein kinases (MAPKs) was determined by Western blotting. Microglia-mediated neuroinflammation was evaluated using a BV-2 microglia-N2a neuron transwell co-culture. RESULTS: PN extract and its component, gallic acid, decreased LPS-induced the mRNA expression of interleukin-6 (IL-6) and inducible nitric oxide synthase (iNOS), as well as IL-6 protein levels in both microglial monoculture and coculture systems. This was accompanied by a reduction in neurodegeneration triggered by microglia in N2a neurons with increased neuronal integrity markers (ßIII tubulin and tyrosine hydroxylase (TH)). These effects were caused by the ability of PN extract to inhibit extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) activation. CONCLUSION: This is the first study to show that PN extract inhibits neurodegeneration in LPS-activated BV-2 microglia by targeting ERK signaling activity.

Phikud Navakot Modulates the Level of Pro-Inflammatory Mediators and the Protein Expression of SOD1 and 2 and the Nrf2/HO-1 Signaling Pathway in Rats with Acute Myocardial Infarction.

Phikud Navakot (PN) is nine major herbs in a famous traditional Thai recipe namely "Yahom Navakot" used to treat cardiovascular disorders. This study investigated the cardioprotective effects of PN formula on isoproterenol-induced myocardial infarction (IMI) in Sprague-Dawley rats. Forty-five rats were randomly divided into nine groups (n = 5 per group): the control, the IMI, the IMI + propranolol, the control or the IMI + PN formula (PN ethanolic extract at doses of 64, 127, or 255 mg/kg) by oroesophageal gavage for 28 days. The ST segment and serum troponin T levels were significantly increased in IMI rats. PN did not eliminate tissue necrosis, infiltration of inflammatory cells, or interstitial edema in IMI rats. All doses of PN decreased (p < 0.001) serum TNF-α and IL-6 levels. PN (127 and 255 mg/kg) up-regulated (p < 0.05) heme oxygenase (HO)-1 expression, whereas PN (255 mg/kg) significantly increased superoxide dismutase (SOD) 1 and 2 expression, compared with IMI rats. Nuclear factor erythroid 2-related factor 2 (Nrf2) and HO-1 expression significantly increased in IMI rats and IMI rats that received PN. PN formula possesses potential anti-inflammatory and antioxidant properties by modulating the levels of TNF-α, IL-6 and antioxidant enzymes. Our study reveals a novel cardioprotective effect of PN in IMI rats through the Nrf2/HO-1 signaling.

Cardioprotection of Atractylodes lancea against Hypoxia/ Reoxygenation-Injured H9c2 Cardiomyoblasts.

Background: Atractylodes lancea (Thunb) DC has been widely used as traditional medicine in many countries including Thailand for the treatment of fever, common cold and sore throat. Objective: To evaluate the cardioprotective effects of Atractylodes lancea extracts against hypoxia/reoxygenation (HR)-injured H9c2 cardiomyoblasts. Material and Method: For cytotoxic determination, the H9c2 cells were incubated with the ethanolic extract of Atractylodes lancea (AL-E) and water extract (AL-W) at the concentrations between 0.01-1 mg/mL in normoxia for 6, 18, 24, and 48 h. Cell viability were determined by MTT assay and observed cellular morphology under phase contrast microscopy. In a timecourse study of HR model on H9c2 cardiomyoblasts, the cells were exposed to hypoxic condition at various time points (0.5, 1, 2, 4, 6, and 8 h) before 24 h reoxygenation. According to more than 90% of cell death, 6 h exposure to hypoxia was used for cardioprotective evaluation throughout the present study. Cell viability, DNA condensation by Hoechst 33342, and protein expression of ERK1/2, p-ERK1/2 and HO-1 by western blot analysis were investigated. Results: Incubation of AL-E and AL-W at concentrations up to 1 mg/mL for 24 h showed no toxicity on H9c2 cells. Exposure of the H9c2 cells to HR showed a time-dependent decrease in cell viability. Treatment of both AL-E and AL-W (0.05 and 0.1 mg/mL) showed protection against cell death and cellular shrinkage as well as inhibited DNA condensation. AL at the same concentrations increased the expression of ERK1/2, p-ERK1/2 and HO-1, when compared to HR-injured cells. Conclusion: AL protected HR-damaged H9c2 cells by inhibiting cell death, cellular shrinkage and DNA condensation, which was partially through restoring ERK1/2, p-ERK1/2, and HO-1 expression. The present results are beneficial use of AL as alternative medicine.

Effects of Phikud Navakot Extract on Myocardial Ischemia/Reperfusion Injury in Rats.

BACKGROUND: Phikud Navakot (PN) is a set of nine medicinal plants and the main ingredient of "Yahom Navakot", a traditional Thai herbal formula for treatment of cardiovascular symptoms. OBJECTIVE: To investigate the cardioprotective effects of PN on myocardial ischemia/reperfusion (IR) in male Sprague Dawley rats. MATERIAL AND METHOD: Rats were randomly divided into 7 groups: sham, IR, and IR orally pretreated with PN (10, 50, 100, 200, and 400 mg/kg B W)for 7 days. After treatment, IR induction was performed by left coronary artery (LCA) ligation for 30 min, followed by reperfusion for 24 h. At the end of the experiment, blood was collected for hematological and biochemical parameters, and hearts were immediately removed for histopathological examination and Western blot analysis. RESULTS: IR induction caused ST elevation in the electrocardiogram and an increase in serum troponin I (TnI), confirming myocardial damage. In addition, histopathological changes of ischemic myocardium showed inflammation, infiltration, and edema. Oral administration of PN (10, 50, 100, 200, and 400 mg/kg BW) for 7 days prior to IR simulation showed no change on serum TnI and histopathology ofcardiac tissues, when compared to IR group. However Western blot analysis showed that IR rats pretreated with PN (10 mg/kg BW) significantly increased (p < 0.05) pERK/ERK ratio, meanwhile pretreated with PN (50-200 mg/kg BW) up-regulated (p < 0.05) the protein expression of HO-1, when compared with IR group. CONCLUSION: The present study implied that 7-day pretreatment of PN failed to protect cardiac tissues against IR injury induced by LCA ligation. Investigation at molecular level found however that PN up-regulated the expression of protective proteins pERK/ERK ratio and HO-1 in cardiac tissues, suggesting molecular mechanism of PN in cardioprotection against IR injury.

Effect of Short-Term Oral Administration of Phikud Navakot in Rats.

BACKGROUND: Phikud Navakot (PN), composed of nine herbs and used as a main component of Yahom Navakot, is used in traditional Thai medicine against dizziness and fainting. OBJECTIVE: To investigate the effects of PN on blood pressure, heart rate (HR), and antioxidant properties on male Sprague Dawley rats. MATERIAL AND METHOD: All rats were weighted everyday in the morning, after that, PN (10, 50, 100, 200 and 400 mg/kg BW) were given oroesophageal feeding for seven days. Systolic blood pressure (SBP), diastolic blood pressure (DBP), mean arterial pressure (MAP), and HR were measured once per two days. At the end of the experiment, the blood was taken for determination of biochemical and hematological parameters, and lipid peroxidation in serum. The heart was immediately removed for Western blot analysis. RESULTS: SBP DBP and MAP of rats were transiently increased after 1 day of PN (100 mg/kg BW) treatment. Meanwhile, HR did not change throughout the experiment. PN (400 mg/kg BW) significantly increased (p< 0.05) the percentage of neutrophils in blood after 7 days of administration. PN treatment has no effect on biochemical parameters and peroxidation of lipid. In addition, ingestion of PN (100 mg/kg BW) significant increased (p < 0.05) HO-1 expression, but did not change ERK1/2 and Bax/Bcl-2 ratio when compared with the control group. CONCLUSION: The results may possibly support the use of PN for prevention and/or alleviation of cardiovascular disorders, caused by reactive oxygen species. However, long-term treatment of PN has to be further studies.

Cissus quadrangularis ethanol extract upregulates superoxide dismutase, glutathione peroxidase and endothelial nitric oxide synthase expression in hydrogen peroxide-injured human ECV304 cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Cissus quadrangularis has been widely used in traditional medicine for the treatment of hemorrhoid. However, the detailed mechanism of antioxidant defense of C. quadrangularis in endothelial cells under oxidative stress remains unclear. AIM OF THE STUDY: The present study aims to elucidate the protective role of ethanol extract of C. quadrangularis (CQE) including its constituents, quercetin and resveratrol, on hydrogen peroxide (H(2)O(2))-injured human umbilical vein endothelial ECV304 cells. MATERIALS AND METHODS: Viability, genotoxicity and protein expression of ECV304 cells were analyzed by MTT, alkaline comet and Western blot, respectively. Production of intracellular reactive oxygen species (ROS) was determined using dichlorofluorescein fluorescence dye. RESULTS: After exposing cells to CQE containing quercetin and resveratrol, DNA damage was not observed. CQE including quercetin and resveratrol significantly attenuated ROS in H(2)O(2)-injured ECV304 cells in a concentration-dependent manner. The protein expression of superoxide dismutase (Cu/Zn-SOD, Mn-SOD), glutathione peroxidase (GPx) and endothelial nitric oxide synthase (eNOS) increased in the cells treated with CQE, quercetin or resveratrol prior to H(2)O(2) exposure, as compared with control. CONCLUSIONS: The results provide a molecular mechanism of C. quadrangularis, which could be partially related to quercetin and resveratrol, in restoring ROS in endothelial cells through the upregulation of Cu/Zn-SOD, Mn-SOD, GPx and eNOS.

Apoptotic effects of satratoxin H is mediated through DNA double-stranded break in PC12 cells.

Satratoxin H is an important air- and food-borne mycotoxin, which has been implicated in human health damage. Satratoxin H is known to induce apoptosis as well as genotoxicity in PC12 cells. In the present study, we further investigated the mechanism of apoptotic effects of satratoxin H with focus on caspase-3 and poly-ADP-ribose polymerase (PARP) pathway. We also examined whether it induces DNA damage in PC12 cells. In the cells treated with satratoxin H, caspase-3 was cleaved in a time-dependent manner. Furthermore, satratoxin H induced cleavage of PARP, one of the downstream molecules of caspase-3. The cleavage was inhibited by SB203580, a p38 MAPK inhibitor, or SP600125, a JNK inhibitor. Satratoxin H, however, had no effect on expression levels of Bax and Bcl-2. Furthermore, the micronucleus assay revealed that satratoxin H induced chromosome break. Also, satratoxin H increased the level of phosphorylation of histone H2A, indicating that it caused DNA double-stranded breaks in PC12 cells. Meanwhile, no genotoxicity was detected with any of treatments carried out in the alkaline comet assay. These results imply that satratoxin H induces genotoxicity by DNA double-stranded break. Our results suggest a considerable potential for the genotoxic risk associated with the presence of satratoxin H.

Effects of Phikud Navakot extract on vascular reactivity in the isolated rat aorta.

The aim of the present study is to investigate the effect of standardized Phikud Navakot extract (NVKE) on aortic rings from male Sprague Dawley rats. Changes in tension were measured using an isometric force transducer and recorded on the PowerLab recording system. Vasorelaxant effect of NVKE was examined in the presence of indomethacin (10 microM), N(G)-nitro L-arginine methyl ester (L-NAME, 300 microM), methoxamine (0.1-300 microM), carbachol (1 nanoM-30 microM), or sodium nitroprusside (0.1 nanoM-10 microM). The results showed that NVKE (1-300 microg/mL) caused vasorelaxation in a concentration-dependent manner with a pEC50 value of 4.27 +/- 0.24 and R(max) of 67.7 +/- 13.9%. Pretreatment with indomethacin or L-NAME did not affect NVKE-induced vasorelaxation. However, co-incubation of indomethacin and L-NAME significantly reduced (p < 0.05) vasorelaxation to NVKE (100 microg/mL). Pre-treatment with NVKE significantly decreased (p < 0.05) endothelium-dependent relaxations to carbachol (R(max) = 52.90 +/- 14.3%), but not to sodium nitroprusside. Moreover contractions to methoxamine were unaffected after pretreatment with NVKE. The present study suggested that NVKE decreased vasorelaxation to carbachol in the rat aorta, which may exert at least against muscarinic receptors. These findings support the use of Phikud Navakot, a major ingredient of Yahom Navakot, against dizziness and fainting.

Stability of barakol under hydrolytic stress conditions and its major degradation product.

The aim of the present study was to investigate the stability of barakol, an anxiolytic constituent extracted from leaves of Senna siamea (Lam.) Irwin & Barneby (syn. Cassia siamea Lam.), under the International Conference on Harmonisation suggested conditions using HPLC with photodiode array detection. Extensive degradation of barakol was found to occur under alkaline conditions through base-catalyzed hydrolysis. Mild degradation of barakol was observed under thermal and oxidative stress while it was stable under acidic conditions. The reaction rate constants (Kobs) of barakol degradation under alkaline conditions at pHs 12 and 13 were 3.0x10(-5) and 9.6x10(-3) min(-1), respectively. The activation energy according to the Arrhenius plot was calculated to be 26.9+/-3.3 kcal/mol at pH 13 and temperatures between 12 and 51 degrees C. The major degradation product of barakol under both alkaline and thermal stress conditions was characterized by LC-MS and NMR as cassiachromone.

Satratoxin H generates reactive oxygen species and lipid peroxides in PC12 cells.

Satratoxin H, a mycotoxin, is thought to induce apoptosis of PC12 cells through the activation of p38 mitogen-activated protein kinase (MAPK) and c-Jun N-terminal kinase (JNK) in a glutathione (GSH)-sensitive manner. The present study was undertaken to further elucidate the mechanism by which satratoxin H induces cell death in PC12 cells. Satratoxin H caused apoptosis of PC12 cells within 24-h, as determined by DNA fragmentation and flow cytometric analysis. Satratoxin H increased reactive oxygen species (ROS) production and lipid peroxidation, as determined by malondialdehyde formation. These effects were attenuated by incubation of cells with GSH, suggesting that satratoxin H-induced increase in apoptosis of serum-deprived PC12 cells may be partially mediated through the generation of ROS.

Involvement of reactive oxygen species and stress-activated MAPKs in satratoxin H-induced apoptosis.

Satratoxins, members of the trichothecene mycotoxin family, have been known to be harmful to health. However, the mechanisms underlying the toxicity still remain unclear. The present study is undertaken to elucidate the mechanisms of the satratoxin H-induced cytotoxicity in PC12 cells. Satratoxin H caused cytotoxicity, which was reflected from apoptosis determined by chromatin staining and flow cytometry. Satratoxin H stimulated the phosphorylation of extracellular signal-regulated kinase (ERK), p38 mitogen-activated protein kinase (MAPK) and c-Jun N-terminal kinase (JNK). Pre-incubation with SB203580, a p38 MAPK inhibitor, or SP600125, a JNK inhibitor, but not PD98059, an ERK inhibitor, reduced satratoxin-induced cytotoxicity. Co-incubation of cells with glutathione, N-acetyl-L-cysteine or glutathione reductase inhibited cytotoxicity and the phosphorylation of p38 MAPK induced by satratoxin H. Our data suggest that satratoxin H-induced apoptosis in PC12 cells is dependent on the activation of p38 MAPK/JNK and the increase in reactive oxygen species.