Novel diallyldisulfide analogs ameliorate cardiovascular remodeling in rats with L-NAME-induced hypertension

Diallyldisulfide (DADS), an active principle of garlic (Allium sativum) is known for its antihypertensive properties. The present study was designed to evaluate the effect of novel DADS analogs, against L-NAME induced hypertension in Wistar rats. The daily administration of L-NAME (50mg/kg) for six weeks along with DADS analogs (20mg/kg) significantly decreased the elevated systolic blood pressure (SBP) and the activity of angiotensin converting enzyme (ACE) and also inhibited the decline in nitrite/nitrate (NO(x)) concentrations and cyclic guanosine monophosphate (cGMP) levels. Adverse changes such as lipid peroxidation, protein damage and a decrease in the levels of antioxidant enzymes, were rectified after the administration of DADS analogs. Oral administration of DADS analogs preserved the expression of endothelial nitric oxide synthase (eNOS). The ability of the DADS analogs to inhibit L-NAME induced hypertension was compared with Enalapril (15mg/kg), which was taken as a standard. The DADS analogs prevented L-NAME-induced cardio toxicity, which was also reflected at the microscopic level indicative of its cardio protective effects. DADS analogs induced vasorelaxation was completely abolished by the removal of the endothelium or by pre-treatment with L-NAME, an inhibitor of nitric oxide synthase. DADS analogs inhibited the calcium influx induced by phenylephrine (0.3μM) and high K(+) (60mM) and this effect was completely abolished by pretreatment of L-NAME. Taken together, our results show that the DADS analogs induce vasorelaxation and have antihypertensive properties, which may be mediated through activation of eNOS.     

Thromboxane prostanoid receptor activation impairs endothelial nitric oxide-dependent vasorelaxations: The role of Rho kinase

Activation of thromboxane prostanoid (TP) receptors causes potent vasoconstriction, which contributes to increased vascular tone and blood pressure. The present study examined the hypothesis that stimulation of TP receptor impaired endothelial nitric oxide-mediated vasorelaxation via a Rho kinase-dependent mechanism. The common carotid arteries of Sprague-Dawley rats were isolated and suspended in myograph for measurement of changes in isometric tension. The production of nitric oxide in primary cultured aortic endothelial cells was assayed with an imaging technique and phosphorylated levels of endothelial NOS were determined by Western blot analysis. 9,11-dideoxy-11α,9α-epoxy-methanoprostaglandin F_2α (U46619) inhibited isoprenaline-induced relaxations in rings with or without endothelium. Treatment with Rho kinase inhibitors, Y27632 (2 μM) or HA 1077 (10 μM) prevented the effect of U46619 only in rings with endothelium while protein kinase C inhibitors were without effect. Rho kinase inhibitors did not affect isoprenaline-induced relaxations in endothelium-intact rings treated with L-NAME or 1H-[1,2,4]oxadizolo[4,3-a]quinoxalin-1-one (ODQ). Isoprenaline stimulated rises in nitric oxide (NO) production in cultured rat endothelial cells. The increased NO production was inhibited by U46619 (100 nM) and this effect was prevented by treatment with Y27632 but unaffected by the absence of extracellular calcium ions. U46619 attenuated isoprenaline-stimulated phosphorylation of eNOS, which was sensitive to inhibition by Y27632 and HA 1077. U46619-mediated effects were abolished by TP receptor antagonist, S18886 and the TP receptor was present in endothelial cells. The present results demonstrate that Rho kinase activation is likely to be the primary mechanism that underlies the U46619-stimulated TP-receptor-mediated inhibition of endothelial NO production and subsequent endothelium-dependent relaxations to isoprenaline.     

不同的机制介导β肾上腺受体激动产生的冠状动脉和股动脉血管舒张

目的：研究β肾上腺素受体激动产生的猪冠状动脉和股动脉血管扩张是否通过不同的机制介导。方法：利用器官组织浴血管环法观察冠状动脉和股动脉在给予不同浓度的异丙肾上腺素（ISO）后血管张力的变化,以及去除血管内皮,一氧化氮合成酶（NOS）抑制,β1和／或β2受体阻断后对其的影响。结果：无论冠状动脉或股动脉ISO均产生浓度依赖的血管舒张。去除内皮,NOS抑制,β2受体阻断不影响ISO产生的冠状动脉血管舒张,而完全消除了股动脉的血管舒张,但消除了冠状动脉的血管舒张。结论：ISO产生的猪冠状动脉血管舒张通过β1受体介导,不通过L－精氨酸／一氧化氮通路,而在股动脉通过β2受体和L－精氨酸／一氧化氮通路。     

In vitro Antioxidant Studies of Fruits of Artemisia nilagirica (Clarke) Pamp

Antioxidant potential of fruits of Artemisia nilagirica was studied using different in vitro models like 1,1-diphenyl-2-picryl hydrazyl, 2,2-azinobis-(3-ethylbenzothizoline-6-sulphonate), nitric oxide, superoxide, hydroxyl radical and lipid peroxidation. Both the ethanol and aqueous extracts of A. nilagirica fruits at 500 μg/ml showed maximum scavenging activity (89.33% and 89.14%) in quenching 1,1-diphenyl-2-picryl hydrazyl radical. The ethanol extract showed better scavenging activity (69.78%) of 1,1-diphenyl-2-picryl hydrazyl radical followed by the scavenging of nitric oxide radical (73.25%) compared to aqueous extract. In contrast, hydroxyl and superoxide radicals were effectively scavenged by aqueous extract. Total antioxidant capacity of ethanol and aqueous extracts at 500 μg/ml concentration was found to be 56.21 and 62.78 mg ascorbic acid equivalents, respectively. However, both the extracts showed only moderate lipid peroxidation inhibition activity. They were also found to contain considerable total phenols and flavonoids suggesting their role as an effective free radical scavenger. These findings suggest that phenolics and flavonoids in the fruits provide substantial antioxidant activity.     

Evaluation of In Vitro Antioxidant Potential of Cordia retusa

The present study was carried out to investigate the antioxidant potential, total flavonoid and phenolic content in extracts of aerial parts of Cordia retua (Vahl.) Masam. The samples such as ethyl acetate and ethanol extracts were tested using six in vitro models such as 2,2-diphenyl-1-picrylhydrazyl, nitric oxide radical, iron chelating, hydroxyl radical, superoxide radical scavenging activity and total antioxidant activity to evaluate the in vitro antioxidant potential of C. retusa by spectrophotometrically. Total flavonoid and phenolic content in samples were estimated using aluminum chloride colorimetric and Folin-Ciocalteu method. The results were analyzed statistically by the regression method. Half maximal inhibitory concentration (IC₅₀) of the ethanol extract was found to be 596 μg/ml for DPPH, 597 μg/ml for nitric oxide radical, 554 μg/ml for iron chelating, 580 μg/ml for hydroxyl radical, 562 μg/ml for superoxide radical and 566 μg/ml for total antioxidant capacity. Furthermore, the total flavonoid content and total phenolic content of the ethanol extract were found to be 2.71 mg gallic acid equivalent per gram of extract and 1.86 mg quercetin equivalent per gram of extract, respectively. In all the testing, a significant correlation existed between concentrations of the extract and percentage inhibition of free radicals. The results of the present comprehensive analysis demonstrated that C. retusa possess potent antioxidant activity, high flavonoid and phenolic content. The antioxidant property may be related to the polyphenols and flavonoids present in the extract. These results clearly indicated that C. retusa is effective against free radical mediated diseases as a natural antioxidant.     

Possible mechanism of acute effect of ethanol on intestinal IgA expression in rat

The purpose of this study was to investigate the possible mechanism of acute effect of ethanol on IgA expression in rat intestine. To this end, adult female Wistar rats showing diestrus day 1 were treated with (a) ethanol (2 or 4 g/kg, i.p.); (b) N omega-nitro-L-arginine-methyl ester (L-NAME), which inhibits the activity of all isoforms of nitric oxide synthase, (30 mg/kg, s.c.) followed by ethanol 3 h later; and (c) L-NAME (30 mg/kg, s.c.) followed by saline 3 h later. Saline-injected and untreated rats were used as controls. The animals were sacrificed 0.5 h after ethanol administration. Intestinal expression of IgA was evaluated by both immunohistochemistry and Western immunoblotting. Morphometric analysis showed that acute ethanol treatment increased the number of IgA-immunoreactive cells in a dose-dependent manner. Pretreatment with L-NAME abolished this action of alcohol. Injection of L-NAME followed by saline had no influence on the number of IgA+cells. The results, obtained by Western immunoblotting, paralleled our immunohistochemical findings. Taken together, these data suggest that acute effect of ethanol on intestinal IgA might be mediated by endogenous nitric oxide.     

Role of endothelial adenosine receptor-mediated vasorelaxation in ethanol-induced hypotension in hypertensive rats

Our previous findings showed that chronic ethanol feeding lowers blood pressure in spontaneously hypertensive rats. The present study investigated the role of the adenosine receptor-endothelial nitric oxide (NO) pathway in the hypotensive response to ethanol. Changes in blood pressure were evaluated in radiotelemetered pair-fed rats receiving liquid diet with or without ethanol (2.5% or 5%, w/v) for 12 weeks. The vasorelaxant activity of the adenosine analogue 5'-N-ethylcarboxamidoadenosine (NECA) in isolated aortic rings obtained from ethanol and control rats were evaluated. Ethanol (2.5% and 5%) lowered blood pressure in a dose-dependent manner. The hypotension started at week 1, reached its maximum at week 4 and remained so thereafter. In aortas with intact endothelium, NECA (10(-10) to 10(-4) M) produced a concentration-dependent relaxation of the phenylephrine-precontracted aortas. Compared with control rats, ethanol (2.5% and 5%) caused significant and concentration-related increases in NECA responses. This effect of ethanol was attenuated by the adenosine receptor antagonist 8-sulfophenyltheophylline and the nitric oxide synthase inhibitor N(G)-monomethyl-L-arginine (L-NMMA). Further, endothelium denudation abolished the ethanol-evoked enhancement of NECA responses. The vasorelaxant responses to acetylcholine or sodium nitroprusside in aortic rings were not influenced by ethanol. In conclusion, the present findings suggest that chronic ethanol enhances the NO-dependent vasorelaxant responses to adenosine receptor activation and this may explain, at least partly, the mechanism of the hypotensive effect of ethanol in spontaneously hypertensive rats.     

Pharmacological investigations of Punica granatum in glycerol-induced acute renal failure in rats

Objective:

The present study was designed to investigate the ameliorative potential and possible mechanism of hydroalcoholic extract of flowers of P. granatum in glycerol-induced acute renal failure (ARF) in rats.

Materials and Methods:

The rats were subjected to rhabdomyolytic ARF by single intramuscular injection of hypertonic glycerol (50% v/v; 8 ml/kg) and the animals were sacrificed after 24 hours of glycerol injection. The plasma creatinine, blood urea nitrogen, creatinine clearance, and histopathological studies were performed to assess the degree of renal injury.

Results:

Pretreatment with hydroalcoholic extract of flowers of P. granatum (125 and 250 mg/kg p.o. twice daily for 3 days) significantly attenuated hypertonic glycerol-induced renal dysfunction in a dose-dependent manner. BADGE (Bisphenol-A-diglycidyl ether) (30 mg/kg), a peroxisome proliferator-activated receptor (PPAR)-γ antagonist, and N(omega)-nitro-l-arginine-methyl ester (L-NAME) (10, 20, and 40 mg/kg), nitric oxide synthase inhibitor, were employed to explore the mechanism of renoprotective effects of Punica granatum. Administration of BADGE (30 mg/kg) and L-NAME (40 mg/kg) abolished the beneficial effects of P. granatum in glycerol-induced renal dysfunction.

Conclusion:

Hydroalcoholic extract of flowers of P. granatum has ameliorative potential in attenuating myoglobinuric renal failure and its renoprotective effects involve activation of PPAR-γ and nitric oxide-dependent signaling pathway.     

17-beta-oestradiol-induced vasorelaxation in vitro is mediated by eNOS through hsp90 and akt/pkb dependent mechanism

1. The L-arginine-NO pathway has been implicated in the vasorelaxant effect of 17-beta-oestradiol. Here we have addressed the involvement of two distinct activation steps of endothelial nitric oxide synthase (eNOS) in the 17-beta-oestradiol-induced vasorelaxant effect on rat aortic rings. 2. Rat aortic rings contracted with phenylephrine (PE) 1 microM relaxed in a concentration related fashion to 17-beta-oestradiol water soluble cyclodextrin-encapsulated (E2) only when endothelium was present. The pure anti-oestrogen of E2 receptor ICI 182,780 (20 microM) significantly inhibited E2-induced vasorelaxation. 3. Geldanamycin (10 microM), a specific inhibitor of heat shock protein 90 (hsp90) and N(omega)-nitro-L-arginine-methyl ester (L-NAME, 100 microM), a nitric oxide synthase inhibitor, significantly inhibited E2-induced vasorelaxation. 4. Incubation of rat aortic rings up to 6 h with LY 294002 (25 microM), a specific inhibitor of PI(3)K akt/pkb pathway reduced E2-induced vasorelaxation. 5. Incubation of rat isolated aorta with E2, induced prostacyclin (PGI(2)) release. PGI(2) levels, measured as 6-keto PGF(1alpha), were abolished by ibuprofen (10 microM), both L-NAME and GA did not influence basal or E2-stimulated PGI(2) confirming the specificity of these two compounds on eNOS pathway. 6. In conclusion, we demonstrate that E2 interaction with its receptor is followed by a vasorelaxant effect in rat aortic rings mediated by eNOS activation through both hsp90 and akt/pkb dependent mechanisms.     

Endothelial nitric oxide modulates perivascular sensory neurotransmission in the rat isolated mesenteric arterial bed

1. A possible role of nitric oxide (NO) as a modulator of capsaicin-sensitive sensory neurotransmission in blood vessels was investigated in the rat isolated mesenteric arterial bed. 2. Electrical field stimulation (EFS) of methoxamine-preconstricted mesenteric beds elicited frequency-dependent vasorelaxation mediated by capsaicin-sensitive sensory nerves. N(G)-nitro-L-arginine methyl ester (L-NAME, 10 and 300 microM) and 7-nitroindazole (7-NI, 100 microM), inhibitors of nitric oxide synthase (NOS), augmented sensory neurogenic vasorelaxation. D-NAME (300 microM), 6-aminoindazole (100 microM) and N(omega)-propyl-L-arginine (50 nM), a selective inhibitor of neuronal NOS, were without effect. The effect of 10 microM L-NAME was reversed by L-arginine (1 mM), the substrate for NOS. 3. L-NAME (300 microM) and 7-NI (100 microM) had no significant effect on vasorelaxations to calcitonin gene-related peptide (CGRP), the principal motor neurotransmitter of capsaicin-sensitive sensory nerves in rat mesenteric arteries, or to capsaicin, indicating a prejunctional action. The inhibitors of NOS had no effect on vasorelaxation to forskolin, but augmented vasorelaxation to sodium nitroprusside (SNP). 4. Removal of the endothelium augmented sensory neurogenic vasorelaxation, but did not affect vasorelaxation to CGRP, indicating a prejunctional action of endothelial NO. 5. In the absence of endothelium, L-NAME (300 microM) inhibited, and 7-NI (100 microM) caused no further augmentation of sensory neurotransmission. 6. SNP (100 nM), a nitric oxide donor, attenuated sensory neurogenic relaxations to EFS. 7. In rat isolated thoracic aortic rings, L-NAME (100 microM) and 7-NI (100 microM) attenuated concentration-dependent relaxations to acetylcholine. 8. These data show that NO modulates sensory neurotransmission evoked by EFS of the rat isolated mesenteric arterial bed, and that when NO synthesis is blocked sensory neurogenic relaxation is augmented. The source of NO is the vascular endothelium.     

盐酸关附甲素对大鼠血管舒缩功能的影响及其机制

目的研究盐酸关附甲素(guanfu base-A,GFA)对大鼠离体胸主动脉舒缩功能的影响,并探讨其可能机制。方法将SD大鼠胸主动脉分离并制成血管环,分成内皮完整组和去内皮组,采用离体血管环实验,观察GFA对基础状态,氯化钾(KCl)预收缩及苯肾上腺素(phenylephrine,PE)预收缩的血管舒张功能的影响,并结合不同抑制剂及无钙液处理,探讨其舒张血管的可能机制。结果累积浓度的GFA(10-8～10-4mol·L-1)对基础状态或KCl预收缩的有无内皮的血管环的张力均无明显影响(P>0.05);对PE(10-6mol·L-1)预收缩内皮完整的血管有浓度依赖性舒张作用,而与PE预收缩的去内皮组相比,从10-7mol·L-1开始差异有显著性(P<0.01)。一氧化氮合酶抑制剂L-NAME、鸟苷酸环化酶抑制剂亚甲蓝及环氧合酶抑制剂吲哚美辛孵育后,均能明显抑制GFA的扩血管作用(P<0.01);经无钙液及GFA处理后,胸主动脉对PE的反应性降低(P<0.01)。结论 GFA对大鼠胸主动脉的舒张作用主要通过两种途径:内皮依赖性舒张作用的机制主要与NO-GC-cGMP途径及激活环氧合酶有关;非内皮依赖性舒张机制主要与抑制内钙释放有关,与门控钙通道引起的外钙内流无关。     

Enhancement of phenylephrine-induced contraction in the isolated rat aorta with endothelium by H2O-extract from an Oriental medicinal plant Leonuri herba.

Leonuri herba (I-mu-ts'ao, the Chinese motherwort) is an ancient Chinese traditional herb. Although the pharmacological effects of extracts of Leonuri herba have been shown in platelets and uteri, the effect on the vascular system has not been determined. In the present study, we investigated the effects of extracts of Leonuri herba on the contraction of the isolated rat aorta. Although the H20-extract (0.3-3 mg/ml) by itself showed a limited effect, the extract enhanced phenylephrine-induced contraction of the aorta with endothelium, but not without endothelium. The H20-extract, like N(G)-nitro-L-arginine methyl ester (L-NAME, an inhibitor of nitric oxide synthase), significantly inhibited the relaxation induced by acetylcholine in the aorta with endothelium. The inhibitory effect of H20-extract on the relaxation decreased by co-addition with 1 mM L-arginine. The vasoconstrictive effect of H20-extract was not due to leonurine, which is a constituent in Leonuri herba and shows uterotonic activity. Intravenous injection of the H20-extract (1.5 mg/kg) to rats caused an increase in blood pressure for 5 min, like L-NAME (1.35 mg/kg). These findings suggest that there is a component(s) in Leonuri herba, which shows a vasoconstrictive activity in rat aorta in vitro and in vivo and has similar pharmacological profile to that of L-NAME.     

Idoxifene causes endothelium-dependent,nitric oxide-mediated vasorelaxation in male rats

Selective estrogen receptor modulators are estrogen-like compounds that lack the deleterious effects of estrogen. The present study was designed to determine whether idoxifene, a new selective estrogen receptor modulator, may have a vasodilatory effect on aortic vessels from male animals, and if so, to investigate the mechanism (i.e., endothelium-independent, direct vasorelaxation vs. endothelium-dependent, nitric oxide mediated vasorelaxation) by which idoxifene may exert its vasodilatory effect. Superior mesenteric arterial rings from adult male Sprague-Dawley rats were suspended in Krebs-Henseleit ring baths. Rings were contracted with 50 nM U-46619 (9,11-epoxymethano-PGH(2)), a thromboxane A(2) mimetic. Cumulative dose-response vasorelaxation to idoxifene (0.01 to 3 microM) was studied in the presence and absence of 200 microM N(omega)-nitro-L-arginine methyl ester (L-NAME, a nitric oxide synthase (NOS) inhibitor). The results obtained from idoxifene were compared with those from 17beta-estradiol. Our experimental results demonstrated that addition of idoxifene to superior mesenteric arterial rings isolated from male rats resulted in a dose-dependent vasorelaxation in the range of 0.1 microM (minimal vasodilatory concentration) to 3 microM (maximal vasodilatory concentration). Pre-treatment with L-NAME to block nitric oxide (NO) production virtually abolished idoxifene-induced vasodilatation, indicating that idoxifene caused an NO-mediated vasorelaxation in vessels from male animals. Addition of 17beta-estradiol also resulted in an endothelium-dependent vasorelaxation in aortic rings from male rats. However, these vessels were 30-fold less sensitive to 17beta-estradiol than to idoxifene in their vasorelaxation responses. Taken together, these results demonstrate that selective estrogen receptor modulators are superior to traditional estrogen in their vascular protection and may thus have potential therapeutic use in protection against cardiovascular disease, especially in male patients.     

Influences of the endothelium and hypoxia on neurogenic transmission in the isolated pulmonary artery of the rabbit.

1. The effects of nitric oxide (10(-6) M), N omega-nitro-L-arginine methylester (L-NAME, 10(-4) M, an inhibitor of nitric oxide synthase), endothelium removal, hypoxia and selective alpha-adrenoceptor antagonists on responses to nerve electrical field-stimulation (EFS) were studied in the rabbit isolated pulmonary artery. 2. EFS induced frequency-dependent contractions which were abolished by prazosin (alpha 1-adrenoceptor antagonist) and unaffected by rauwolscine (alpha 2-adrenoceptor antagonist). EFS-induced responses were potentiated by L-NAME and inhibited by nitric oxide. The effect of L-NAME was reversed by the presence of L-arginine (2 x 10(-4) M), which had no effect on its own. In the presence of L-NAME, the EFS-induced responses were reduced by rauwolscine and the residual responses were abolished by prazosin. 3. Removal of the vascular endothelium increased the maximum contractile response to EFS but did not inhibit the ability of L-NAME to potentiate contractile responses to EFS. 4. Hypoxia inhibited the contractile response to EFS. This effect of hypoxia was also seen in the presence of L-NAME and in endothelium rubbed preparations. 5. In conclusion, the endothelium modulates EFS-induced contractions in the rabbit pulmonary artery. The contraction induced by EFS was inhibited by nitric oxide, but potentiated by the nitric oxide-synthase inhibitor, L-NAME. The effect of L-NAME was not mediated solely through the endothelium and revealed involvement of alpha 2-adrenoceptors in EFS-induced contraction. Hypoxia inhibited neurogenic responses in rabbit isolated pulmonary arteries.     

Mesenteric vascular bed responsiveness in bile duct-ligated rats: roles of opioid and nitric oxide systems.

Changes in vascular responsiveness are proposed as the basis for some of the cardiovascular complications in cholestasis. Cholestasis is also associated with accumulation of endogenous opioid peptides and evidence of overproduction of nitric oxide (NO). The possible role of NO or opioid system in cholestasis-induced mesenteric vascular bed responsiveness was investigated. Bile duct-ligated and sham-operated rats were treated for 6 days with either normal saline, naltrexone, an opioid antagonist (20 mg/kg/day) or L-NAME (N(omega)-nitro-L-arginine methyl ester), a nitric oxide synthase inhibitor (3 mg/kg/day). After 7 days, the superior mesenteric artery was cannulated and the mesenteric vascular bed was perfused according to the McGregor method. Baseline perfusion pressure of the mesenteric vascular bed was decreased in bile duct-ligated compared to sham-operated animals. ED(50) of phenylephrine-induced vasoconstriction was increased, but vasoconstriction R(max) was not different in the vascular bed of bile duct-ligated rats and of sham-operated ones. Acetylcholine-induced vasorelaxation was impaired in bile duct-ligated rats (increased ED(50) and decreased vasorelaxation R(max)). Sodium nitroprusside-induced vasorelaxation was not different between bile duct-ligated and sham-operated rats, implying that the smooth muscle components of vasorelaxation were intact. Chronic treatment with L-NAME partially restored both the acetylcholine-induced vasorelaxation and phenylephrine-induced vasoconstriction response in bile duct-ligated rats. Naltrexone treatment also partially restored the acetylcholine-induced vasorelaxation and phenylephrine-induced vasoconstriction in bile duct-ligated rats. There is impaired acetylcholine-induced vasorelaxation in cholestatic rats, probably due to a defect in endothelial function. This study also provided evidence for the involvement of increased opioidergic tone and NO overproduction in cholestasis-induced vascular hyporesponsiveness.     

Betulinic acid ameliorates endothelium-dependent relaxation in L-NAME-induced hypertensive rats by reducing oxidative stress

Zizyphi Spinosi semen (ZSS) is one of the most widely used traditional Chinese herbs with protective effects on the cardiovascular system. It is not clear whether betulinic acid (BA), the key active constituent of ZSS, has beneficial cardiovascular effects on N(ω)-nitro-L-arginine methyl ester hydrochloride (L-NAME)-induced hypertensive rats. The objective of this study was to investigate the effect of BA on endothelium-dependent vasorelaxation in isolated aortic rings from L-NAME-induced hypertensive rats and its underlying mechanisms. Male Sprague-Dawley rats were injected with L-NAME (15 mg/kg/d, i.p.) for 4 weeks to induce hypertension. After treatment with L-NAME for 2 weeks, rats with mean blood pressure >120 mm Hg measured by tail-cuff method were considered hypertensive and then injected with BA (0.8, 4, 20 mg/kg/d, i.p.) for the last 2 weeks. The effect of BA on the tension of rat thoracic aortic rings was measured in an organ bath system. The levels of nitric oxide (NO), reactive oxygen species (ROS), and the activity of superoxide dismutase (SOD) and nitric oxide synthase (NOS) in aortas were assayed. We found that BA (0.1-100 μM) evoked a concentration-dependent vasorelaxation in endothelium-intact normal rat aortic rings, which was significantly attenuated by pretreatment with L-NAME (100 μM) or methylene blue (MB, 10 μM), but not by indomethacin (10 μM). Pretreatment with EC(50) (1.67 μM) concentration of BA enhanced the acetylcholine (ACh)-induced vasorelaxation, which was also markedly reversed by both L-NAME and MB. The blood pressure in hypertensive rats increased to 135.22±5.38 mm Hg (P<0.01 vs. control group), which was markedly attenuated by high dose of BA. The ACh-induced vasorelaxation in hypertensive rat aortic rings was impaired, which was markedly improved by chronic treatment with BA (20 mg/kg/d) for 2 weeks. The increase of ROS level and the decrease of NO level, SOD and eNOS activities in hypertensive rat aortas were all markedly inhibited by BA. These results indicate that BA decreased blood pressure and improved ACh-induced endothelium-dependent vasorelaxation in L-NAME-induced hypertension rats, which may be mediated by reducing oxidative stress and retaining the bioavailability of NO in the cardiovascular system.     

The role of nitric oxide in the reversal of hemorrhagic shock by oxotremorine.

In the present study, the effect of the nitric oxide synthase inhibitor, N(G)-nitro-L-arginine methylester (L-NAME), on the antishock actions of oxotremorine was investigated in rats subjected to hemorrhagic shock under urethane anesthesia. L-citrulline production in the AV3V region, as an indicator of nitric oxide (NO) synthesis, was assayed by high-performance liquid chromatography (HPLC) with fluorescent detection throughout the experiment. The rats were pretreated with either intravenous (i.v.) physiological saline or L-NAME (2.5 mg/kg) before bleeding. L-NAME potentiated the reversal of hypotension by oxotremorine (25 microg/kg, i.v.). However, oxotremorine either alone or in combination with L-NAME did not produce any significant change in 60-min survival rate at this low dose. Analysis of microdialysis samples collected from the AV3V region showed that L-citrulline concentration increased during bleeding and that this increase was abolished by L-NAME pretreatment. These results may suggest that nitric oxide production contributes to hypotension in rats bled to shock since nitric oxide levels in the AV3V region increased in response to bleeding and nitric oxide synthase (NOS) inhibition abolished this increase and potentiated the oxotremorine-induced reversal of hypotension.     

Evaluation and Comparison of the In Vitro Cytotoxic Activity of Withania somnifera Methanolic and Ethanolic Extracts against MDA-MB-231 and Vero Cell Lines

Withania somnifera Dunal (WS), commonly known as Ashwagandha in India, belongs to the family Solanaceae. It is extensively used in most of the Indian herbal pharmaceuticals and nutraceuticals. In the current study, the in vitro cytotoxic activity of methanolic, ethanolic, and aqueous extracts of WS stems was evaluated using cytometry and the MTT assay against the MDA-MB-231 human breast cancer cell line. Methanolic and ethanolic extracts of WS showed potent anticancer activity on the MDA-MB-231 human breast cancer cell line, whereas the aqueous extract did not exhibit any significant activity at 100 µg/ml. The percentage viability of the cell lines was determined by using the Trypan blue dye exclusion method. Cell viability was reduced to 21% and 0% at 50 and 100 µg/ml of the methanolic extract, respectively, as compared to 19% and 0% at 50 and 100 µg/ml for the ethanolic extract and 37% at 100 µg/ml in sterile Milli-Q water after 48 hours of treatment. Methanolic and ethanolic extracts of WS were shown to possess IC₅₀ values of 30 and 37 µg/ml, respectively, by the MTT assay and cytometer-based analysis, with the methanolic extract being more active than the other two. On the other hand, methanolic and ethanolic extracts of WS did not exhibit any significant in vitro activity against the normal epithelial cell line Vero at 50 µg/ml. HPLC was carried out for the analysis of its phytochemical profile and demonstrated the presence of the active component Withaferin A in both extracts. The methanolic and ethanolic extracts of Withania should be studied further for the isolation and characterization of the active components to lead optimization studies.     

Evaluation of Parmotrema reticulatum Taylor for Antibacterial and Antiinflammatory Activities

Lichens produce variety of secondary metabolites including depsides, depsidones and dibenzofurans having multifunctional activity in response to external environmental condition. Present study provides evidence for in vitro antibacterial and in vivo antiinflammatory activity of acetone and ethanol extracts of Parmotrema reticulatum. In vitro antibacterial activity was investigated against gram positive and gram negative bacteria. Cotton pellet-induced granuloma, xylene-induced ear swelling, carragennan-induced edema, histamine-induced and carboxymethylcellulose sodium-induced leukocyte emigration in mice models were used to quantify the antiinflammatory activity. Acetone and ethanol extracts were showed antibacterial activity against Bacillus subtilis (minimal inhibitory concentration: 100 and 150 μg/ml) and Staphylococcus aureus (minimal inhibitory concentration: 100 and 200 μg/ml), Escherichia coli (minimal inhibitory concentration: 200 and 250 μg/ml), and Pseudomonasa eruginosa (minimal inhibitory concentration: 200 and 300 μg/ml). Acetone extract was inhibited edema significantly at 200 mg/kg with xylene, cotton pellet, carragennan and histamine induced edema in vivo models. Ethanol extract was found effective at dose of 300 mg/kg with all in vivo antiinflammatory models. The results showed significant (P<0.01) antiinflammatory effects at 200 and 300 mg/kg dose of acetone and ethanol extracts, respectively, which can be concluded that significant activity may be due to presence of flavanoids in ethanol extract and usnic acid in acetone extract.