Insights into the formulation properties, biocompatibility, and permeability of poorly water-soluble methoxyflavones with PEG400 and propylene glycol.

Herein, thermal and non-thermal techniques were used to elucidate the putative physical and chemical interactions between poorly water-soluble Kaempferia methoxyflavones and PEG400/propylene glycol. Additionally, the biocompatibility of methoxyflavone-glycol solutions was evaluated using Caco-2 cells whereas the absorptive transport was investigated by measuring the apparent permeability coefficient (P app) of the methoxyflavones and transepithelial electrical resistance (TEER) of the Caco-2 cell monolayer. Data from differential scanning calorimetry, Fourier-transform infrared (FTIR), and proton nuclear magnetic resonance (1H NMR) spectroscopic analysis revealed physico-chemical compatibility between the three methoxyflavones and PEG400/propylene glycol. Furthermore, PEG400 and propylene glycol solutions of the methoxyflavones were shown to be compatible with Caco-2 cells at pharmacologically effective concentrations. In vitro transport studies across the Caco-2 cell monolayer revealed high P app values of 24.07 × 10-6 to 19.63 × 10-6 cm s-1 for PEG400 solutions of the methoxyflavones. The TEER values of the Caco-2 cell monolayers indicated that the increased drug transport was partly due to increased tight junction openings, but without compromising the epithelial barrier integrity. The good pharmaceutical and biocompatibility profiles, as well as improved transport of the methoxyflavones in PEG400 and propylene glycol solutions, are suggestive of the worthiness of this approach for further consideration pertaining to the development of these drugs into oral liquid dosage forms.

Decreased body-fat accumulation and increased vasorelaxation to glyceryl trinitrate in middle-aged male rats following six-weeks consumption of coconut milk protein

Abstract We investigated whether coconut milk protein (CMP) contributes to the beneficial effects of coconut milk consumption on cardiovascular health markers previously found in middle-aged rats. CMP was isolated and precipitated from dried fresh coconut milk, then gavaged (1 g/kg) to middle-aged male rats for six weeks; control rats received distilled water. Compared to controls, CMP caused decreased body fat and lipid accumulation in liver cells and the platelet count. CMP did not affect basal blood pressure or heart rate in anesthetized rats. Vascular responsiveness to phenylephrine, DL-propargylglycine (PAG), acetylcholine or sodium nitroprusside was unaffected, but vasorelaxation to glyceryl trinitrate (GTN) increased. Effects of ODQ on vasorelaxation to GTN were similar in both groups. Expression of blood vessel eNOS, CSE and sGC was normal. The cyclic guanosine monophosphate (cGMP) level of CMP-treated rats was normal but addition of GTN increased cGMP and NO concentration more in CMP-treated rats than in controls, an effect unaltered by addition of diadzin. Taken together, CMP appears partially responsible for the improvement in cardiovascular health markers caused by coconut milk in middle-aged male rats

Effect of consumption of whole egg and egg fractions on cardiovascular disease factors in adult rats.

BACKGROUND: While eggs are a low-cost source of protein, rich in macro- and micronutrients, the association of egg intake and cardiovascular disease (CVD) remains controversial. This study investigated the effect of egg consumption on CVD parameters. Eggs were boiled, separated into four fractions (whole egg, 50% yolk-reduced whole egg, egg yolk and egg white) and then freeze-dried. The different egg fractions or distilled water (control) were orally gavaged to adult male Wistar rats at 1 g kg-1 rat body weight, each day for 8 weeks, following which basal blood pressure, heart rate, complete blood cell count, blood biochemistry, body fat and liver cell lipid accumulation were determined. The vascular functions of isolated thoracic aorta were studied using classical pharmacological techniques. RESULTS: In comparison to the control group, none of the egg fractions affected body weight, food intake, plasma glucose or lipid profile. The yolk group experienced increased plasma alkaline phosphatase and creatinine levels, while egg white caused decreased plasma cholesterol and blood urea nitrogen. Whole egg and egg yolk increased blood pressure and mean hemoglobin concentration and the yolk increased liver lipid accumulation. Egg white decreased the white blood cell count and body fat lipids. No changes were found in basal heart rate or vascular functions in any of the groups. CONCLUSIONS: Consumption of whole egg or egg yolk at the dosage given caused hypertension, with impairment of liver and kidney functions following the intake of yolk alone. However, egg white is beneficial for the cardiovascular system as it decreased plasma cholesterol and body fat accumulation. © 2020 Society of Chemical Industry.

Enhancing oral absorption of poorly water-soluble herb (Kaempferia parviflora) extract using self-nanoemulsifying formulation.

Kaempferia parviflora, a medicinal herb, treats hypertension and promotes longevity with good health and well-being. Its bioactive component is poorly soluble in water, resulting in poor absorption. This study aimed to enhance the bioavailability of K. parviflora dichloromethane (KPD) extract using a self-nanoemulsifying drug delivery system (SNEDDS). KPD was dissolved in diethylene glycol monoethyl, polyoxyl-35 castor oil and caprylic/capric glyceride, and clear yellow SNEDDS solution was obtained. The methoxyflavone markers were used for content and dissolution analysis. Solid SNEDDS was prepared by stepwise mixing of KPD using a mortar and pestle (1:1 ratio) with five solid carriers: Aerosil® 200, Florite® RE, Neusilin® US2 (NEUS), Fujicalin®, and Neusilin® UFL2. The USP apparatus II with simulated gastric fluid USP (SGF without pepsin, pH 1.2) was used in order to perform the in vitro dissolution. The methoxyflavones dissolution at 60 min from KPD, SEDDS, and SNEDDS/NEUS were approximately 16, 92, and 73%, respectively. The pharmacokinetic profiles of methoxyflavones for oral administration were studied using Wistar rats; the areas under the curve of SNEDDS/NEUS (1.77-fold) and SNEDDS (5.38-fold) were significantly higher than that of KPD. The developed formulations showed good stability after storage for 6 months under accelerated and normal conditions.

6 weeks consumption of pure fresh coconut milk caused up-regulation of eNOS and CSE protein expression in middle-aged male rats

Coconut milk (CCM) has been an important cooking ingredient in the Asia-Pacific region since ancient time. Due to its high content of saturated fatty acids, it has been considered atherogenic. We have tested if chronic consumption of fresh coconut milk by middle-aged male rat affects vascular function, plasma glucose and lipid profiles. Compared to control, CCM caused lower maximal contraction to phenylephrine of thoracic aortic rings and increased relaxation to acetylcholine that was abolished by N G-nitro-L-arginine (L-NA) or disruption of the endothelium. DL-propargylglycine caused slight increase in baseline tension of L-NA treated aortic rings of CCM-treated rats and produced higher contractile response of the aortic rings to low concentrations of phenylephrine. The aortic eNOS- and cystathionine-γ-lyase(CSE) proteins expression of the CCM-treated rats were also higher than in controls. Except for lower fasting plasma glucose there were no changes in blood chemistry for the CCM treated rats. CCM consumption caused up-regulation of eNOS and CSE protein expression which resulted in increased production of NO and H2S from the blood vessels with attenuation of vasocontraction to phenylephrine and increased relaxation to acetylcholine. These novel benefits may be expected to reduce the development of cardiovascular risk factors in the aging rat

Improved dissolution of Kaempferia parviflora extract for oral administration by preparing solid dispersion via solvent evaporation.

Kaempferia parviflora, a plant in the family Zingiberaceae, has been used in Thai traditional medicines for treating hypertension and promoting longevity with good health and well-being. However, its limited aqueous solubility and low dissolution restrict its bioavailability. The aim of the study was therefore to improve the dissolution rate of K. parviflora extracted with dichloromethane (KPD) by solid dispersions. Different water-soluble polymers were applied to improve dissolution of KPD. The solid dispersions in different ratios were prepared by solvent evaporation method. Only hydroxypropyl methylcellulose (HPMC) and polyvinyl alcohol-polyethylene glycol grafted copolymer (PVA-co-PEG) could be used to produce homogeneous, powdered solid dispersions. Physical characterization by scanning electron microscopy, hot stage microscopy, differential scanning calorimetry and powder X-ray diffractometry, in comparison with corresponding physical mixtures, showed the changes in solid state during the formation of solid dispersions. Dissolution of a selected marker, 5,7,4'-trimethoxyflavone (TMF), from KPD/HPMC and KPD/PVA-co-PEG solid dispersions was significantly improved, compared with pure KPD. The dissolution enhancement by solid dispersion was influenced by both type and content of polymers. The stability of KPD/HPMC and KPD/PVA-co-PEG solid dispersions was also good after 6-month storage in both long-term and accelerated conditions. These results identified that the KPD/HPMC and KPD/PVA-co-PEG solid dispersions were an effective new approach for pharmaceutical application of K. parviflora.

Increased vascular eNOS and cystathionine-γ-lyase protein after 6 weeks oral administration of 3, 5, 7, 3', 4'-pentamethoxyflavone to middle-aged male rats.

Effects of treatment of middle-aged male rats with 3, 5, 7, 3', 4'-pentamethoxyflavone (PMF) on vascular and perivascular adipose tissue (PVAT) functions and blood chemistry were investigated. Rats received PMF (22 mg/kg), orally or vehicle, twice a day for 6 weeks. The PMF-treated rats had lower serum glucose, higher HDL-C levels, but no change in other parameters. Thoracic aortic and mesenteric rings of PMF treated rats produced lower maximal contraction to phenylephrine that was normalized by NG-nitro-L-arginine (L-NA) or endothelial removal. The aortic- and mesenteric rings of the PMF treated rats showed improved relaxation to acetylcholine, but not to glyceryl trinitrate, and had higher eNOS protein. DL-propargylglycine (PAG) caused greater increase in the baseline tension of the PMF-treated aortic ring and higher contraction to low concentrations of phenylephrine. PVAT lowered the contractile response of the L-NA pretreated aortic rings to phenylephrine for both groups, but PAG had no effect. The cystathionine-γ-lyase (CSE) protein of the thoracic rings, but not of the PVAT, shows increased expression after PMF treatment. Overall, PMF treatment of middle aged rats appeared to increase production of NO and H2S from the blood vessels by upregulating the expression of eNOS and CSE. PMF also decreased fasting serum glucose and increased HDL-C levels, with no toxicity to liver and kidney functions. Thus, PMF is a novel compound for possible use as a health product to prevent and/or to reduce the development of diabetes type II and/or cardiovascular disease.

Effects of 6 weeks oral administration of Phyllanthus acidus leaf water extract on the vascular functions of middle-aged male rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Leaves of Phyllanthus acidus (PA) have been used in Thai traditional medicine for the treatment of hypertension. We have previously shown that chronic treatment of a PA water extract to middle-aged male rats caused a lowering of the body and serum lipids, two of the parameters that are implicated in cardiovascular disease. AIM OF THE STUDY: To investigate if chronic treatment of middle-aged male rats with a PA water extract affected the perivascular (aortic) adipose tissue (PVAT) and/or their vascular functions MATERIALS AND METHODS: Fresh leaves of PA were extracted with water and orally gavaged to the middle-aged male rats for 6 weeks. Vascular functions were studied in vitro using isolated thoracic aorta with and without PVAT, and mesenteric rings in Krebs Heinseleit solution with results recorded with a Polygraph or a Myograph system. The amount of blood vessel eNOS and CSE (cystathionine-γ-lyase) expression was measured by Western blotting. RESULTS: PA treatment caused a lower maximal contractile response to phenylephrine (Phe) of the endothelium-intact aortic ring than that of the control group. This effect was abolished by N(G)-nitro-l-arginine (l-NA) or by denudation of the endothelium. dl-propargylglycine (PAG, H2S inhibitor) and TEA (Ca(2+)-activated K(+) channel blocker), but not glybenclamide (ATP-activated K(+) channel blocker), caused a similar increase in the baseline of the endothelium-intact aortic ring in the presence of l-NA in both the PA-treated and control aortic rings. This effect sequentially resulted in a greater contractile response of the aortic rings of both groups to Phe. Glybenclamide also caused a similar increase in the maximal contraction of the endothelium-intact blood vessels with l-NA to both groups. PAG, TEA or glybenclamide did not modify the phenylephrine C-R curves for either group of the PVAT-endothelium-intact aortic rings preincubated with l-NA. The CSE levels of the thoracic aorta and at the PVAT were not different between the PA-treated and the control group. Relaxation of the Phe-precontracted thoracic aortic ring to acetylcholine, but not to glyceryl trinitrate, was higher for the PA-treated than for the control aortic rings and this effect was abolished by l-NA. The mesenteric rings of the PA treated group showed a lower sensitivity on the contractile response to Phe than that of the control group, and this effect was abolished by l-NA. Vasodilatation to acetylcholine, but not to glyceryl trinitrate, of the PA treated-mesenteric ring was more sensitive than that of the control group and this effect was abolished by l-NA. The expression of eNOS by the PA treated thoracic aorta and the mesenteric arteries was higher than the control group. These results demonstrated that chronic treatment with a PA water extract to middle-aged rats affected their vascular functions by increasing the nitric oxide production from the endothelial cells and also modulated the responsiveness of the thoracic aortic- and mesenteric rings to phenylephrine and acetylcholine.

Enhanced dissolution and oral bioavailability of nifedipine by spontaneous emulsifying powders: effect of solid carriers and dietary state.

The objective of this study was to prepare spontaneous emulsifying powder (SEP) for improving dissolution and enhancing oral bioavailability of a poorly water-soluble drug, nifedipine (NDP). In order to investigate the effects of solid carrier properties, such as surface area and pore size, and a concurrent food intake on absorption of NDP in rats, different SEP formulations were prepared by adsorbing liquid spontaneous emulsifying formulation (SEF), composing of polyoxyl 35 castor oil, caprylic/capric glyceride and diethylene glycol monoethyl ether at a ratio of 1:1:8, onto various solid carriers (i.e., silica (FS), porous calcium silicate (PCS) and porous silicon dioxide). The solid characterization by scanning electron microscopy, differential scanning calorimetry and powder X-ray diffraction revealed the absence of crystalline NDP in the formulations. SEP also demonstrated excellent spontaneous emulsification properties similar to SEF. The droplet size of emulsions formed after dilution was less than 200 nm. The solid carriers (particularly PCS) had significant and positive effect in drug dissolution; the mean dissolution time of SEP containing PCS was considerably improved. SEP also provided a good stability after storage in accelerated and long-term conditions for 6 months. The bioavailability study resulted in enhanced values of C(max) and AUC for SEP formulations, when tested in both fasted and fed rats. Furthermore, comparing the AUC in fasted and fed rats, NDP powder exhibited a significant food effect. The difference in bioavailability of NDP in fed compared to fasted state can be avoided by using SEP.

Effects of Kaempferia parviflora rhizomes dichloromethane extract on vascular functions in middle-aged male rat.

ETHNOPHARMACOLOGICAL RELEVANCE: In Thai traditional medicine, rhizomes of Kaempferia parviflora (KP) have been used for treating hypertension and for the promotion of longevity with good health and well being. Ageing is one of the most important risk factors for development of cardiovascular disease. To investigate whether a 6 weeks oral administration of a dichloromethane extract of fresh rhizomes of Kaempferia parviflora (KPD) had any effects on vascular functions, on the accumulation of lipid, as well as on any signs of gross organ toxicity in middle-aged rats. MATERIALS AND METHODS: Fresh rhizomes of Kaempferia parviflora were first macerated twice with 95% ethanol to remove the dark color before extracting three times with 100% dichloromethane. The dichloromethane extract was evaporated under reduced pressure to obtain the dried Kaempferia parviflora dichloromethane extract (KPD). The rats were orally administered with the KPD at a dosage of 100mg/kg body weight, or with the same volume of the vehicle (tween 80, 0.2g: carboxy-methylcellulose sodium, 0.2g: distilled water 10 ml) once or twice a day for 6 weeks. Vascular functions were studied on isolated thoracic aorta and the mesenteric artery. The vascular eNOS enzyme was measured by Western blot analysis. Blood chemistry was measured by enzymatic methods. Liver cell lipid accumulation was measured using oil red O staining. RESULTS: A 6 weeks treatment of KPD once a day had no significant effects on any of the studied parameters. When the KPD was given twice a day, the contractile responses to phenylephrine of the thoracic aorta and mesenteric artery were lower than the vehicle control group, and this effect was abolished by N(G)-nitro-l-arginine or by removal of the vascular endothelium. Vasorelaxation to acetylcholine, but not to glyceryl trinitrate, by the thoracic aortic and mesenteric ring precontracted with phenylephrine was higher from the KPD treated rats than those from the vehicle control groups. Western blot analysis showed a higher quantity of thoracic- and mesenteric-eNOS protein obtained from the KPD treated rats. In addition, the body weight, serum glucose and triglycerides levels, visceral and subcutaneous fat, as well as liver lipid accumulation were all significantly decreased in the KPD treated rats compared to those of the vehicle control. No differences were found between the KPD treated-, and the vehicle-control for animal food intake, internal organ weight, serum ALP, SGOT, SGPT, BUN and creatinine levels, serum cholesterol, HDL-C and LDL-C levels, nor total blood cell counts. CONCLUSIONS: The chronic oral administration of KPD extract, to middle aged rats, caused a decrease in vascular responsiveness to phenylephrine with an increase in the acetylcholine induced vasorelaxation, due to an increase in nitric oxide production from their blood vessels. The extract also caused a decrease in visceral and subcutaneous fat, fasting serum glucose and triglyceride levels and liver lipid accumulation, with no changes to liver and kidney functions or to total blood cell counts. It is possible that these KPD extracts could be developed as a health product for mid-aged humans to reduce obesity, diabetes type II and cardiovascular disease.

Six weeks oral gavage of a Phyllanthus acidus leaf water extract decreased visceral fat, the serum lipid profile and liver lipid accumulation in middle-aged male rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Advancing age is associated with an increased accumulation of visceral fat and liver lipid which is then responsible for an age-related risk for cardiovascular disease. Looking after ourselves well with suitable micronutrients could prevent disease or prolong our healthy cardiovascular functions. In Thai traditional medicine, leaves of Phyllanthus acidus (PA) have been used for many purposes including as an antihypertensive agent and to provide relief from a headache caused by hypertension. We aimed to investigate the effects of a chronic oral administration of PA extracts to middle-aged (12-14 months) rats on their body weight, food intake, body fats, liver and kidney functions, fasting blood glucose and lipid profiles, liver lipid accumulation and on blood pressure. MATERIALS AND METHODS: Three different kinds of PA extracts were used: (1) a PA water extract, (2) a heated PA water extract, and (3) an n-butanol fraction of the PA water extract, prepared from fresh leaves of Phyllanthus acidus. The rats were orally gavaged with the three PA extracts at 1.0 g/kg body weight or, as a control, with distilled water once a day for 6 weeks. Fasting blood sugar, lipid profile and ALP, SGOT, SGPT, BUN and creatinine levels were measured by enzymatic methods. Liver lipid accumulation was measured using oil red O staining on fresh thin cryostat liver tissue sections. The animal basal blood pressure and heart rate were measured in anesthetized rats via a common carotid artery using a polygraph. RESULTS: Results showed that after 6 weeks of treatment using gavaged heated PA extract and PA n-butanol extract there were no changes in any of the parameters studied. However, the initial PA water extract caused a slight decrease in the animal body weight with no change in food intake. No changes were observed in the liver and kidney functions (serum ALP, SGOT, SGPT, BUN and creatinine did not change), nor did the fasting blood sugar or triglyceride levels differ significantly. Serum cholesterol, HDL and LDL levels, as well as visceral and subcutaneous adipose tissue and liver lipid accumulation were significantly decreased compared to that of the control group. There were no differences found in the basal systolic and diastolic blood pressure and the basal heart rate between the PA water extract treatment and the control group. CONCLUSIONS: These results indicated that the PA water extract had an effect on lipid metabolisms that resulted in a decrease of the serum lipid profile, visceral and subcutaneous fat, as well as on liver lipid accumulation in middle-aged rats. The active component that is responsible for these effects is likely to be a water soluble substance(s) and is heat labile. As a consequence of these beneficial effects of the PA water extract, it would be a good choice for further development for use as a nutraceutical or health product to prevent and/or to slow down the development of obesity and/or cardiovascular disease.

Crude extract and purified components isolated from the stems of Tinospora crispa exhibit positive inotropic effects on the isolated left atrium of rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Tinospora crispa has been used in folkloric medicine for the control of blood pressure. We previously found that an extract of Tinospora crispa and its constituents effect the heart rate and blood pressure in anesthetized rats. AIM OF THE STUDY: The aim was to investigate the effects and mechanisms of the Tinospora crispa extract and bioactive components on the rat isolated left atria. MATERIALS AND METHODS: Air-dried stems of Tinospora crispa were extracted with water, followed by partitioning with chloroform, ethyl acetate, and finally by n-butanol. The n-butanol soluble material was concentrated and dried under reduced pressure and lyophilized to obtain a crude powder (Tinospora crispa extract). The active components of Tinospora crispa extract were separated by column chromatography and preparative HPLC. The effects and mechanisms of the n-butanol extract and the bioactive purified components (adenine, uridine, adenosine, salsolinol, tyramine, higenamine, syringin, (-)-litcubinine, borapetoside A, borapetoside B, borapetoside D and borapetoside E) were studied in isolated left atria from normal and reserpinized rats. RESULTS: Tinospora crispa extract caused an increase in the force of contraction of the electrical field stimulated left atrium. This effect was inhibited by propranolol, atenolol, ICI-118,551, phentolamine and atropine. The positive inotropic effect on the reserpenized isolated left atrium of the Tinospora crispa extract was significantly inhibited by propranolol, atenolol and ICI-118,551. Phentolamine, on the other hand, caused potentiation and the effect was inhibited when propranolol was also added. Higenamine caused an increase in the force of contraction of the electrical field stimulated left atrium and this effect was significantly inhibited by ICI-118,551 and atenolol but not by phentolamine. Reserpine did not significantly shift the concentration-response curve (C-R curve) of the inotropic effect of the higenamine. ICI-118,551 and atenolol caused a parallel shift of the C-R curve to the right of about 8 and 33 fold, respectively. At low concentrations salsolinol caused a slight increase in the force of contraction of the left atrium, but at higher concentrations a decrease was observed. The negative inotropic effect of salsolinol was significantly inhibited by propranolol and atropine. In the reserpinized isolated left atrium, the negative inotropic effect of salsolinol was potentiated and again this effect was significantly inhibited by propranolol and atropine. Tyramine caused a positive inotropic effect, and this effect was inhibited by propranolol or by pretreatment of the rat with reserpine. Adenosine caused a negative inotropic effect, while uridine caused a slight positive inotropic effect on the left atrium. This effect was significantly inhibited by DPCPX. CONCLUSIONS: Crude extract of Tinospora crispa exert a positive inotropic effect on the electrical field stimulated isolated left atria that results from the concerted action of 5 bioactive compounds: higenamine, salsolinol, tyramine, adenosine and uridine. Higenamine, salsolinol (at low concentration) and tyramine acted via the adrenergic receptors to increase the force of the atrial contraction, whereas a high concentration of salsolinol acted indirectly by stimulating the release of acetylcholine. Adenosine and uridine acted via the purinergic pathways to cause negative inotropic effects on the isolated left atria.

Relaxant mechanisms of 3, 5, 7, 3', 4'-pentamethoxyflavone on isolated human cavernosum.

We have investigated effects and mechanisms responsible for the activity of 3, 5, 7, 3', 4'-pentamethoxyflavone (PMF) on isolated human cavernosum. PMF is the major flavone isolated from Kaempferia parviflora claimed to act as an aphrodisiac. PMF caused relaxation of phenylephrine precontracted human cavernosal strips, and this effect was slightly inhibited by N(G)-nitro-l-arginine, a nitric oxide synthase inhibitor, but not by ODQ (soluble guanylate cyclase inhibitor), TEA (tetraethylammonium, blocker of voltage-dependent K(+) channels) or glybenclamide (blocker of ATP-dependent K(+) channels). PMF did not significantly inhibit the relaxant activity of glyceryltrinitrate or acetylcholine on human cavernosal strips precontracted with phenylephrine. In contrast, sildenafil (phosphodiesterase inhibitor) potentiated the relaxant activity of glyceryl trinitrate but not of acetylcholine. In normal Krebs solution with nifedipine (blocker of l-type Ca(2+) channels), or in Ca(2+)-free Krebs solution, PMF caused a further inhibition of human cavernosum contracted with phenylephrine. In human cavernosum treated with thapsigargin (inhibitor of sarcoplasmic reticulum Ca(2+)-ATPase) in Ca(2+)-free medium, PMF suppressed the concentration-response curve of human cavernosum to phenylephrine and a further suppression was found when SKF-96365 (a blocker of store-operated Ca(2+) channels and Y-27632 (inhibitor of Rho-kinase)), but not nifedipine, were added sequentially. Thus, PMF had only a weak effect on the release of nitric oxide, and had no effect as a K(ATP)- or K(Ca) channel opener, a phosphodiesterase inhibitor, a store-operated Ca(2+) channel blocker or a Rho-kinase inhibitor. Therefore, these studies suggest that PMF causes relaxation of human cavernosum through voltage-dependent Ca(2+) channels and other mechanisms associated with calcium mobilization.

Hypotensive and cardio-chronotropic constituents of Tinospora crispa and mechanisms of action on the cardiovascular system in anesthetized rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Tinospora crispa has been used in folkloric medicine for the control of blood pressure. We previously found that an extract of Tinospora crispa stems decreased the mean arterial blood pressure (MAP) with a transient decrease, followed by an increase in the heart rate (HR) in rats. AIM OF THE STUDY: To identify the active components of the Tinospora crispa extract and investigate the mechanisms of action on blood pressure and heart rate in anesthetized rats. MATERIALS AND METHODS: The active components of Tinospora crispa extract were separated by column chromatography and a preparative HPLC. The effects and mechanisms of the active compounds on blood pressure and heart rate were studied in anesthetized, normal and reserpinized rats in vivo. RESULTS: 5 active compounds: adenosine, uridine, salsolinol, higenamine and tyramine were isolated. Adenosine decreased MAP and HR and this effect was inhibited by DMPX (A(2A) adenosine receptor antagonist). Uridine increased MAP and decreased HR and this was inhibited by suramin but not by DMPX. Salsolinol decreased the MAP and HR and this was inhibited by phentolamine but not by ICI-118,551 (ß(2)-adrenoceptor antagonist) or atropine. In reserpinized rats, salsolinol had a hypertensive effect that was inhibited by prazosin and phentolamine, but not by atenolol, and caused an increase in HR that was inhibited by atenolol, but not by prazosin or phentolamine. Higenamine decreased the MAP with an increase in HR. The hypotensive effect was inhibited by ICI-118,551 or atenolol, whereas the increase in HR was not inhibited by ICI-118,551. Atenolol inhibited the increase in HR at a small dosage of higenamine but potentiated it at a higher dosage. In reserpinized rats, a small dosage of higenamine tended to potentiate the effect but at a higher dosage it caused inhibition. ICI-118,551 significantly inhibited this hypotensive effect. Tyramine caused an increase in MAP and HR and these effects almost disappeared in reserpinized rats. CONCLUSIONS: The results demonstrate that these 5 compounds from Tinospora crispa acted in concert on the cardiovascular system of anesthetized rats. Salsolinol, tyramine and higenamine acted via the adrenoreceptors, whereas uridine and adenosine acted via the purinergic adenosine A(2) and P(2) receptors to decrease blood pressure with a transient decrease of HR followed by an increase.

Effects of an n-butanol extract from the stem of Tinospora crispa on blood pressure and heart rate in anesthetized rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Tinospora crispa has been used in folkloric medicine for control of blood pressure, as an antipyretic, for cooling down the body temperature and for maintaining good health. AIM OF THE STUDY: To investigate the effects and mechanisms of action of an n-butanol extract from the stems of Tinospora crispa (T. crispa extract) on blood pressure and heart rate in anesthetized rats. MATERIALS AND METHODS: Air-dried stems of T. crispa were extracted with water, followed by partitioned extract with chloroform, ethyl acetate, and finally by n-butanol. The n-butanol soluble part was evaporated under reduced pressure and lyophilization to obtain a crude dried powder (T. crispa extract). The effects and mechanisms of the T. crispa extract on blood pressure and heart rate were studied in anesthetized normal and reserpinized rats in vivo in the presence of different antagonists. RESULTS: T. crispa extract (1-100 mg/kg, i.v.) caused a decrease in mean arterial blood pressure (MAP) and this effect was inhibited by propranolol, phentolamine, atenolol and/or the ß(2)-antagonist ICI-118,551, but not by atropine or hexamethonium. In reserpinized rats, the T. crispa extract had a dual effect: reduction in hypotensive activity, followed by a small increase in blood pressure. The decrease in MAP in reserpinized rat was slightly potentiated by phentolamine, but inhibited by propranolol or ICI-118,551 only if atenolol and phentolamine were also present. The increase in MAP was potentiated by propranolol and ICI-118,551, but was inhibited by phentolamine. The T. crispa extract had a dual effect on heart rate in the normal rat: a small transient decrease, followed by an increase in heart rate. The positive chronotropic effect of T. crispa extract was inhibited by propranolol, phentolamine and atenolol, but not by ICI-118,551, atropine or hexamethonium. Reserpine potentiated the positive chronotropic effect of the T. crispa extract and this effect was inhibited by propranolol, atenolol and ICI-118,551, but not by phentolamine. CONCLUSIONS: From these results we suggest that T. crispa extract possesses at least three different cardiovascular-active components that act directly via (1) ß(2)-adrenergic receptors to cause a decrease in blood pressure, and ß(1)- and ß(2)-adrenergic receptors to cause an increase in heart rate, (2) α-adrenergic receptors to cause an increase in blood pressure and heart rate, and (3) a non-adrenergic and non-cholinergic pathway to cause a decrease in MAP and heart rate. These findings provide scientific support for the tradition of using this plant to modify the actions of the human cardiovascular system.

Hypotensive activity of an n-butanol extract and their purified compounds from leaves of Phyllanthus acidus (L.) Skeels in rats.

We aimed to investigate the effects, identify the active substances and establish the mechanisms involved in the hypotensive activity of an n-butanol extract from leaves of Phyllanthus acidus (PA extract). PA extract caused a decrease in blood pressure of anesthetized rats that was not modified by atropine or propranolol. PA extract caused a persistent dilatation of thoracic aortic rings preconstricted with either phenylephrine or KCl, and these effects were not modified by LNA or removal of the vascular endothelium. For phenylephrine-preconstricted aortic rings, the dilatory activity of the PA extract was not modified by atropine, propranolol or indomethacin. TEA, glybenclamide or ODQ significantly inhibited the dilatory activity of the PA extract on endothelium-denuded aortic rings. Nifedipine or a Ca(2+)-free medium depressed the aortic rings constrictor response to phenylephrine, and that was further augmented by the PA extract. Adenosine, 4-hydroxybenzoic acid, caffeic acid, hypogallic acid, and kaempferol were isolated from the PA extract. Each caused a decrease in blood pressure and dilatation of the aortic rings. LNA or removal of the endothelium reduced this activity. ODQ and TEA attenuated the vasodilatory activity of adenosine whereas glybenclamide and ODQ attenuated the effect of hypogallic acid. These results suggest that the hypotensive activities of the PA extract is likely the result of the direct action of these five compounds on the blood vessels by stimulating release of nitric oxide from the vascular endothelium, in part through stimulation of soluble guanylate cyclase, and opening of K(ATP) and K(Ca) channels in the vascular smooth muscle.

Cardiovascular effects of tyramine: adrenergic and cholinergic interactions.

The cardiovascular effects of tyramine, and its interactions with propranolol, atenolol, phentolamine, prazosin, yohimbine and atropine, have been investigated in anesthetized rats in vivo and in vitro. Tyramine (i.v.) increased both the mean arterial blood pressure and heart rate. Phentolamine, prazosin or a combination of prazosin and yohimbine caused an inhibition of the hypertensive effect of tyramine, but propranolol, atenolol and/or atropine had no effect. Propranolol added to rats receiving atropine depressed the chronotropic effect of tyramine. However, in non-atropinized animals, the positive chronotropic effect of tyramine was paradoxically enhanced by propranolol, and further enhanced by atropine. A similar result was found with atenolol pre-treated animals. Phentolamine did not alter the effects of propranolol and atropine on heart rate. Prazosin depressed the positive chronotropic effects of tyramine, and this effect was re-stored by pre-treatment of the animals with both prazosin and yohimbine. In the isolated atria, propranolol--in contrast to the in vivo results--inhibited the chronotropic effect of tyramine. Atropine potentiated the positive inotropic effect of isoproterenol and tyramine. Tyramine produced a concentration-dependent contraction of isolated thoracic aortic rings, and this was potentiated by N(G)-nitro-l-arginine, or by removal of the endothelium, but inhibited by phentolamine. The in vivo and in vitro effects of tyramine were not seen in rats which had been treated with reserpine. We conclude that the positive inotropic and chronotropic effects of tyramine in the rat are due to indirect release of transmitter. The results suggest that the paradoxical enhancement by propranolol and atenolol of the chronotropic effect in vivo could be due to tyramine causing increased vagal activity, such that an inhibitory effect of propranolol can only be revealed in animals treated with atropine.

Cardiovascular effects of an n-butanol extract from fresh fruits of Randia siamensis.

Randia siamensis is used in Thai folklore medicine for inducing abortion and controlling blood pressure. The present study investigated the cardiovascular effects of an R. siamensis fruit extract, and mechanisms involved in anesthetized normal and reserpinized rats. R. siamensis (0.4-12 mg/kg) i.v. increased the mean arterial pressure (MAP) and heart rate. Both effects were significantly inhibited by phentolamine (2 mg/kg, i.v.) or propranolol (0.6 mg/kg, i.v.). The combination of phentolamine and propranolol, or reserpine pretreatment, inhibited the positive chronotropic effect with a slight decrease in the MAP. In vitro, R. siamensis (0.001-0.3 mg/ml) increased the rate of beating of the right atrium and the strength of the electrical field-stimulated contraction of the left atrium, both effects were inhibited by propranolol, or with reserpine pretreated rats. R. siamensis (0.01-3 mg/ml) produced a contraction of isolated thoracic aorta, which was potentiated by N(G)-nitro-L-arginine (LNA), or by removal of the vascular endothelium, but inhibited by phentolamine, or reserpine. R. siamensis (0.3-3 mg/ml) caused a relaxation of phenylephrine-preconstricted aortic rings, which was potentiated with reserpine pretreatment, and abolished after removal of the vascular endothelium, or in the presence of LNA. These results suggest that R. siamensis extract exerts both hypertensive and positive chronotropic effects via the alpha- and beta-adrenergic receptors of blood vessels and the heart, due to release of endogenous catecholamines, likely from nerve ending and adrenal medulla. The hypotensive activity results from the release of nitric oxide causing dilatation of the blood vessels. The present data support the folklore therapeutic uses of this plant.

An extract from the medicinal plant Phyllanthus acidus and its isolated compounds induce airway chloride secretion: A potential treatment for cystic fibrosis.

According to previous reports, flavonoids and nutraceuticals correct defective electrolyte transport in cystic fibrosis (CF) airways. Traditional medicinal plants from China and Thailand contain phytoflavonoids and other bioactive compounds. We examined herbal extracts of the common Thai medicinal euphorbiaceous plant Phyllanthus acidus for their potential effects on epithelial transport. Functional assays by Ussing chamber, patch-clamping, double-electrode voltage-clamp and Ca2+ imaging demonstrate activation of Cl- secretion and inhibition of Na+ absorption by P. acidus. No cytotoxic effects of P. acidus could be detected. Mucosal application of P. acidus to native mouse trachea suggested transient and steady-state activation of Cl- secretion by increasing both intracellular Ca2+ and cAMP. These effects were mimicked by a mix of the isolated components adenosine, kaempferol, and hypogallic acid. Additional experiments in human airway cells and CF transmembrane conductance regulator (CFTR)-expressing BHK cells and Xenopus laevis oocytes confirm the results obtained in native tissues. Cl- secretion was also induced in tracheas of CF mice homozygous for Phe508del-CFTR and in Phe508del-CFTR homozygous human airway epithelial cells. Taken together, P. acidus corrects defective electrolyte transport in CF airways by parallel mechanisms including 1) increasing the intracellular levels of second messengers cAMP and Ca2+, thereby activating Ca2+-dependent Cl- channels and residual CFTR-Cl- conductance; 2) stimulating basolateral K+ channels; 3) redistributing cellular localization of CFTR; 4) directly activating CFTR; and 5) inhibiting ENaC through activation of CFTR. These combinatorial effects on epithelial transport may provide a novel complementary nutraceutical treatment for the CF lung disease.

Isoflavone glycosides from Derris scandens.

Five isoflavone glycosides, named derriscandenosides A-E (1-5), were isolated from the stems of Derris scandens, together with ten known compounds comprising one isoflavone, two benzoic acid derivatives, three glucosyl isoflavones and four rhamnosyl-(1-->6)-glucosyl isoflavones. The structures of the glycosides were assigned on the basis of spectroscopic data, especially of the acetate derivatives. Three known rhamnosyl-(1-->6)-glucosyl isoflavones isolated from a crude fraction were retested for hypotensive activity with varying results.