Selection of suitable endogenous reference genes for qPCR in kidney and hypothalamus of rats under testosterone influence.

Real-time quantitative PCR (qPCR) is the most reliable and accurate technique for analyses of gene expression. Endogenous reference genes are being used to normalize qPCR data even though their expression may vary under different conditions and in different tissues. Nonetheless, verification of expression of reference genes in selected studied tissue is essential in order to accurately assess the level of expression of target genes of interest. Therefore, in this study, we attempted to examine six commonly used reference genes in order to identify the gene being expressed most constantly under the influence of testosterone in the kidneys and hypothalamus. The reference genes include glyceraldehyde-3-phosphate dehydrogenase (GAPDH), actin beta (ACTB), beta-2 microglobulin (B2m), hypoxanthine phosphoribosyltransferase 1 (HPRT), peptidylprolylisomerase A (Ppia) and hydroxymethylbilane synthase (Hmbs). The cycle threshold (Ct) value for each gene was determined and data obtained were analyzed using the software programs NormFinder, geNorm, BestKeeper, and rank aggregation. Results showed that Hmbs and Ppia genes were the most stably expressed in the hypothalamus. Meanwhile, in kidneys, Hmbs and GAPDH appeared to be the most constant genes. In conclusion, variations in expression levels of reference genes occur in kidneys and hypothalamus under similar conditions; thus, it is important to verify reference gene levels in these tissues prior to commencing any studies.

Effects of Root Extracts of Eurycoma longifolia Jack on Corpus Cavernosum of Rat.

OBJECTIVE: This study was conducted to investigate the mechanisms of action of Eurycoma longifolia in rat corpus cavernosum. MATERIALS AND METHODS: Tincture of the roots was concentrated to dryness by evaporating the ethanol in vacuo. This ethanolic extract was partitioned into 5 fractions sequentially with hexane, dichloromethane (DCM), ethyl acetate, butanol, and water. The corpus cavernosum relaxant activity of each fraction was investigated. The DCM fraction which showed the highest potency in relaxing phenylephrine-precontracted corpora cavernosa was purified by column chromatography. The effects of the most potent DCM subfraction in relaxing phenylephrine-precontracted corpora cavernosa, DCM-I, on angiotensin I- or angiotensin II-induced contractions in corpora cavernosa were investigated. The effects of DCM-I pretreatment on the responses of phenylephrine-precontracted corpora cavernosa to angiotensin II or bradykinin were also studied. An in vitro assay was conducted to evaluate the effect of DCM-I on angiotensin-converting enzyme activity. RESULTS: Fraction DCM-I decreased the maximal contractions (100%) evoked by angiotensin I and angiotensin II to 30 ± 14% and 26 ± 16% (p < 0.001), respectively. In phenylephrine-precontracted corpora cavernosa, DCM-I pretreatment caused angiotensin II to induce 82 ± 27% relaxation of maximal contraction (p < 0.01) and enhanced (p < 0.001) bradykinin-induced relaxations from 47 ± 8% to 100 ± 5%. In vitro, DCM-I was able to reduce (p < 0.001) the maximal angiotensin-converting enzyme activity to 78 ± 0.24%. CONCLUSION: Fraction DCM-I was able to antagonize angiotensin II-induced contraction to cause corpus cavernosum relaxation via inhibition of angiotensin II type 1 receptor and enhance bradykinin-induced relaxation through inhibition of angiotensin-converting enzyme.

First Report of Eurycoma longifolia Jack Root Extract Causing Relaxation of Aortic Rings in Rats.

Although Eurycoma longifolia has been studied for erectile function, the blood pressure- (BP-) lowering effect has yet to be verified. Hence, this study aims at investigating the BP-lowering properties of the plant with a view to develop an antihypertensive agent that could also preserve erectile function. Ethanolic root extract was partitioned by hexane, dichloromethane (DCM), ethyl acetate, butanol, and water. The DCM fraction, found to be potent in relaxing phenylephrine- (PE-) precontracted rat aortic rings, was further purified by column chromatography. Subfraction DCM-II, being the most active in relaxing aortae, was studied for effects on the renin-angiotensin and kallikrein-kinin systems in aortic rings. The effect of DCM-II on angiotensin-converting enzyme (ACE) activity was also evaluated in vitro. Results showed that DCM-II reduced (p < 0.05) the contractions evoked by angiotensin I and angiotensin II (Ang II). In PE-precontracted rings treated with DCM-II, the Ang II-induced contraction was attenuated (p < 0.05) while bradykinin- (BK-) induced relaxation enhanced (p < 0.001). In vitro, DCM-II inhibited (p < 0.001) the activity of ACE. These data demonstrate that the vasodilatory effect of DCM-II appears to be mediated via inhibition of Ang II type 1 receptor and ACE as well as enhancement of Ang II type 2 receptor activation and BK activity.

Potassium channel openers and prostacyclin play a crucial role in mediating the vasorelaxant activity of Gynura procumbens.

BACKGROUND: Previous studies of Gynura procumbens (G. procumbens) have shown that partially purified fractions of the leaves are capable of lowering the blood pressure of rats by inhibiting angiotensin-converting enzymic activity and causing vasodilatation. The objectives of this study were therefore to further purify the active compounds that exhibited selective effects on blood vessels, determine the mechanism of actions, and to qualitatively analyse the putative compounds present. METHODS: The butanolic fraction (BU) of the crude ethanolic extract was purified using column chromatography to obtain several sub-fractions of different polarities. The in vitro effects of BU and the sub-fractions on vascular tension were subsequently determined using isolated rat thoracic aortic rings. The most potent sub-fraction (F1) alone was then investigated for its mechanisms of the vasorelaxant activity. In another experiment, thin-layer chromatography was used to qualitatively analyse the active compounds found in F1. RESULTS: The BU and the sub-fractions ranging from 10-7 to 10-2 g/ml significantly (p < 0.05) inhibited the sustained tonic contractions induced by phenylephrine and potassium chloride in a concentration-dependent manner with various degree of potency. The most potent sub-fraction (F1) antagonised the calcium-induced vasocontractions (1 x 10-4 - 1 x 10-2 M) in calcium-free with high concentration of potassium as well as in calcium- and potassium-free Krebs-Henseleit solutions. Contractions induced by noradrenaline and caffeine were not affected by F1. The vasorelaxing effect caused by F1 was significantly attenuated with preincubation of potassium channel blockers (glibenclamide and 4-aminopyridine) and prostacyclin inhibitor (indomethacin) while it was not affected by preincubation with tetraethylammonium, l-nitro-arginine methyl esther, propanolol, atropine, oxadiazolo quinoxalin one and methylene blue. The qualitative phytochemical analysis of F1 indicated the presence of flavonoids. CONCLUSION: These results confirm previous findings that G. procumbens causes vasodilatory effects by blocking calcium channels. In addition, the present study further demonstrates that the vasodilatory effect of G. procumbens may also be due to the opening of potassium channels and the stimulation of prostacyclin production. The putative compounds are probably flavonoids in nature.

Gynura procumbens causes vasodilation by inhibiting angiotensin II and enhancing bradykinin actions.

Previous studies showed that Gynura procumbens reduced blood pressure by blocking calcium channels and inhibiting the angiotensin-converting enzyme activity. The present experiments were to further explore the effects and mechanisms of a purer aqueous fraction (FA-I) of G. procumbens on angiotensin I (Ang I)-induced and angiotensin II (Ang II)-induced contraction of aortic rings and also on the bradykinin (BK) effect on cardiovascular system. Rat aortic rings suspended in organ chambers were used to investigate the vascular reactivity of FA-I. Effect of FA-I on BK was studied by in vitro and in vivo methods. Results show that FA-I significantly (P < 0.05) decreased the contraction evoked by Ang I and Ang II. In the presence of indomethacin (10 µM) or N-nitro-L-arginine methyl ester (0.1 µM), the inhibitory effect of FA-I on Ang II-induced contraction of aortic rings was reduced. Besides, FA-I potentiated the vasorelaxant effect and enhanced the blood pressure-lowering effect of BK. In conclusion, FA-I reduced the contraction evoked by Ang II probably via the endothelium-dependent pathways, which involve activation of the release of nitric oxide and prostaglandins. The inhibition of angiotensin-converting enzyme activity by FA-I may contribute to the potentiation of the effects of BK on cardiovascular system.

Gynura procumbens Merr. decreases blood pressure in rats by vasodilatation via inhibition of calcium channels.

INTRODUCTION: Gynura procumbens has been shown to decrease blood pressure via inhibition of the angiotensinconverting enzyme. However, other mechanisms that may contribute to the hypotensive effect have not been studied. OBJECTIVES: To investigate the cardiovascular effects of a butanolic fraction of Gynura procumbens in rats. METHODS: Anaesthetized rats were given intravenous bolus injections of butanolic fraction at doses of 2.5-20 mg/kg in vivo. The effect of butanolic fraction on vascular reactivity was recorded in isolated rat aortic rings in vitro. RESULTS: Intravenous administrations of butanolic fraction elicited significant (p < 0.001) and dose-dependent decreases in the mean arterial pressure. However, a significant (p < 0.05) decrease in the heart rate was observed only at the higher doses (10 and 20 mg/kg). In isolated preparations of rat aortic rings, phenylephrine (1 × 10⁻6 M)- or potassium chloride (8 × 10⁻² M)-precontracted endothelium-intact and -denuded tissue; butanolic fraction (1 × 10⁻6 - 1 × 10⁻¹ g/ml) induced similar concentration-dependent relaxation of the vessels. In the presence of 2.5 × 10⁻³ and 5.0 × 10⁻³ g/ml butanolic fraction, the contractions induced by phenylephrine (1 × 10⁻9-3 × 10⁻5 M) and potassium chloride (1 × 10⁻² - 8 × 10⁻² M) were significantly antagonized. The calcium-induced vasocontractions (1 × 10⁻4-1 × 10⁻²M) were antagonized by butanolic fraction concentration-dependently in calcium-free and high potassium (6×10⁻² M) medium, as well as in calcium- and potassium-free medium containing 1×10⁻6 M phenylephrine. However, the contractions induced by noradrenaline (1 × 10⁻6 M) and caffeine (4.5 × 10⁻² M) were not affected by butanolic fraction. CONCLUSION: Butanolic fraction contains putative hypotensive compounds that appear to inhibit calcium influx via receptor-operated and/or voltage-dependent calcium channels to cause vasodilation and a consequent fall in blood pressure.

Gynura procumbens Merr. decreases blood pressure in rats by vasodilatation via inhibition of calcium channels

INTRODUCTION: Gynura procumbens has been shown to decrease blood pressure via inhibition of the angiotensinconverting enzyme. However, other mechanisms that may contribute to the hypotensive effect have not been studied. OBJECTIVES: To investigate the cardiovascular effects of a butanolic fraction of Gynura procumbens in rats. METHODS: Anaesthetized rats were given intravenous bolus injections of butanolic fraction at doses of 2.5-20 mg/kg in vivo. The effect of butanolic fraction on vascular reactivity was recorded in isolated rat aortic rings in vitro. RESULTS: Intravenous administrations of butanolic fraction elicited significant (p<0.001) and dose-dependent decreases in the mean arterial pressure. However, a significant (p<0.05) decrease in the heart rate was observed only at the higher doses (10 and 20 mg/kg). In isolated preparations of rat aortic rings, phenylephrine (1×10-6 M)- or potassium chloride (8×10-2 M)-precontracted endothelium-intact and -denuded tissue; butanolic fraction (1×10-6-1×10-1 g/ml) induced similar concentration-dependent relaxation of the vessels. In the presence of 2.5×10-3 and 5.0×10-3 g/ml butanolic fraction, the contractions induced by phenylephrine (1×10-9-3×10-5 M) and potassium chloride (1×10-2-8×10-2 M) were significantly antagonized. The calcium-induced vasocontractions (1×10-4-1×10-2 M) were antagonized by butanolic fraction concentration-dependently in calcium-free and high potassium (6×10-2 M) medium, as well as in calcium- and potassium-free medium containing 1×10-6 M phenylephrine. However, the contractions induced by noradrenaline (1×10-6 M) and caffeine (4.5×10-2 M) were not affected by butanolic fraction. CONCLUSION: Butanolic fraction contains putative hypotensive compounds that appear to inhibit calcium influx via receptor-operated and/or voltage-dependent calcium channels to cause vasodilation and a consequent fall in blood pressure.

Inhibition of angiotensin-converting enzyme activity by a partially purified fraction of Gynura procumbens in spontaneously hypertensive rats.

OBJECTIVES: To investigate the hypotensive and angiotensin-converting enzyme (ACE) inhibitory activities of a partially purified fraction (FA-I) of the leaves of Gynura procumbens and to qualitatively analyse the putative compounds present in the fraction. MATERIALS AND METHODS: The hypotensive effect of FA-I was tested in both spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto rats (WKY) by an intravenous administration of 0-10 mg/kg of the FA-I. Administration of captopril (20 microg/kg) served as the control. In vitro 0.0-2.0 mg/ml FA-I was added to a mixture of ACE and hippuryl-L-histidyl-L-leucine and assayed by a modification of the colourimetric method of Hurst and Lovell-Smith. All blood pressure measurements were monitored by the Macintosh MacLab set-up. ACE activity was measured by an in vitro assay in which the enzymatic cleavage of hippuryl-L-histidyl-L-leucine to form histidyl-leucine and hippurate was determined colourimetrically by a cyanuric chloride/dioxane reagent. RESULTS: The FA-I produced a marked dose-dependent reduction in mean arterial pressure (MAP) in SHR and WKY rats, with an ED(50) of 1.09 and 1.05 mg/kg, respectively (p < 0.01). Furthermore, FA-I at 10 mg/kg strongly inhibited the angiotensin I-induced rise in MAP (p < 0.01). This response was comparable to that of captopril at 20 microg/kg. In the in vitro assay, ACE activity was inhibited with an IC(50) of 0.8 mg/ml. The qualitative phytochemical analysis of FA-I indicated the presence of glycoconjugates and peptides. CONCLUSION: These results suggest that the hypotensive effect of G. procumbens may be due, in part, to the glycoconjugated or peptidal substances found in FA-I that exhibit an inhibitory effect on ACE.