In vitro study on α-amylase inhibitory activity of an Indian medicinal plant, Phyllanthus amarus

Objective:

The objective of this study was to evaluate the α-amylase inhibitory activity of different extracts of Phyllanthus amarus against porcine pancreatic amylase in vitro.

Materials and Methods:

The plant extracts were prepared sequentially with ethanol, chloroform, and hexane. Each extract was evaporated using rotary evaporator, under reduced pressure. Different concentrations (10, 20, 40, 60, 80, and 100 μg/mL) of each extract were made by using dimethyl sulfoxide (DMSO) and subjected to α-amylase inhibitory assay using starch azure as a substrate. The absorbance was read at 595 nm using spectrophotometer. Using this method, the percentage of α-amylase inhibitory activity and IC₅₀values of each extract was calculated.

Results:

The chloroform extract failed to inhibit α-amylase activity. However, the ethanol and hexane extracts of P. amarus exhibited appreciable α-amylase inhibitory activity with an IC50 values 36.05 ± 4.01 μg/mL and 48.92 ± 3.43 μg/mL, respectively, when compared with acarbose (IC₅₀value 83.33 ± 0.34 μg/mL).

Conclusion:

This study supports the ayurvedic concept that ethanol and hexane extracts of P. amarus exhibit considerable α-amylase inhibitory activities. Further, this study supports its usage in ethnomedicines for management of diabetes.     

Evaluation of Cynanchum otophyllum glucan sulfate against human immunodeficiency virus and herpes simplex virus as a microbicide agent

Objective:

The root of Cynanchum otophyllum—also known as Qing Yang Sheng—is a traditional ethnical Chinese medicine. The objective of this study was to evaluate in vitro activities and safety of C. otophyllum glucan sulfate (PS20) against Human Immunodeficiency Virus (HIV) and Herpes Simplex Virus (HSV).

Materials and Methods:

Anti-HIV activity was detected with syncytial formation assay and quantitative P24 Enzyme-Linked Immunosorbent Assay (ELISA). Anti-HSV activity was detected with plaque reduction assay; cytotoxicity was tested with MTT colorimetric assay; and anti-bacterial activity was tested with microdilution method. Anti-HIV mechanism was investigated with fusion inhibition, time of addition, and pretreatment.

Results:

The 50% Inhibition Concentration (IC₅₀) of PS20 for HIV-1_IIIB, HIV-_Ada-M, HIV-1_Bal, HSV-I, and -II were 0.26 ± 0.02 mM, 0.46 ± 0.02 mM, 0.90 ± 0.04 mM, 3.45 ± 0.85 μM, and 0.70 ± 0.22 mM, respectively. Selectivity Indices (SI) were 653, 50, 39, 85, and 362, respectively. Studies on anti-HIV mechanism of PS20 showed that the target molecule should be the envelope protein. The 50% Cytotoxicity Concentrations (CC₅₀) of PS20 for HeLa and ME-180 cell lines and human foreskin fibroblast cells was more than 70 μM. The Minimum Inhibitory Concentration (MIC) for vaginal lactobacilli was more than 1000 μM.

Conclusion:

PS20 possesses anti-HIV and HSV effect and low cytotoxicity to epithelium cells and vaginal lactobacilli. It may be considered as a potential microbicide agent for further investigation.     

Effect of erythromycin and rifampicin on monoethylglycinexylidide test

Background:

The dynamic liver function test based on the hepatic conversion of lidocaine to monoethylglycinexylidide (MEGX) provides a direct measure of the actual functional state of the liver. Cytochrome P450 (CYP) 3A4 has been proposed as the main CYP isoform responsible for MEGX formation. The concomitant use of either CYP3A4 inducer rifampicin or CYP3A4 inhibitor erythromycin may influence the results of MEGX test. Hence, the objective of this study was to evaluate the effect of a CYP3A4 inhibitor erythromycin and inducer rifampicin on the MEGX test.

Materials and Methods:

The study included 20 healthy male volunteers whose routine laboratory tests were normal. As per study protocol, MEGX test was carried out in all the participants after an overnight fast. All the participants were given 1 mg/kg lidocaine dose intravenously and MEGX concentration at 30 and 60 min after IV dose was measured using HPLC. These MEGX values served as control values. Ten subjects received 600 mg/day erythromycin orally for six days while remaining ten participants received 600 mg/day rifampicin orally for six days. On the sixth day, MEGX test was carried out two hours after the last dose.

Result:

Rifampicin increased the mean plasma concentration of MEGX₃₀ from 93.94 ± 26.31 to 98.54 ± 24.94 μg/ml (P = 0.085) and MEGX₆₀ from 134.34 ± 35.42 to 136.36 ± 33.14 μg/ml (P = 0.051). Erythromycin lowered the serum concentration of MEGX₃₀ from 101.37 ± 39.39 to 96.67 ± 36.09 μg/ml (P = 0.128) and MEGX₆₀ from 142.52 ± 42.65 to 138.98 ± 40.23 μg/ml (P = 0.159).

Conclusion:

It can be concluded from this study that the MEGX test is not affected by concomitant administration of CYP3A4 modifiers rifampicin and erythromycin.     

Standardized Clitoria ternatea leaf extract as hyaluronidase, elastase and matrix-metalloproteinase-1 inhibitor

Aim:

Plant Clitoria ternatea L. is claimed to possess a wide range of activities including antiinflammatory, local anesthetic and antidiabetic effect, etc. The aim of the present study was to evaluate the wound healing potential of standardized C. ternatea leaf extract in terms of different enzymatic models, which are mostly associated with skin wound.

Materials and Methods:

The methanol extract and fractions were screened for its hyaluronidase, elastase, and matrix metalloproteinase-1 (MMP-1) inhibitory activity compared with standard oleanolic acid. The activity was rationalized through reverse phase high performance liquid chromatography (RP-HPLC) standardization of the extract and fractions with respect to its isolated biomarker taraxerol (yield 5.27% w/w).

Results:

The extract showed significant (P < 0.001) hyaluronidase (IC₅₀ 18.08 ± 0.46 μg/ ml) and MMP-1 (P < 0.05) inhibition, but the elastase inhibition was insignificant (IC₅₀ 42.68 ± 0.46 μg/ml). Among the fractions, ethyl acetate fraction showed significant (P < 0.001) inhibition of hyaluronidase (IC₅₀ 28.01 ± 0.48 μg/ml) and MMP-1 (P < 0.01). The HPLC analysis revealed that the extract and the ethyl acetate fraction are enriched with taraxerol (5.32% w/w and 4.55% w/w, respectively).

Conclusions:

The experiment validated the traditional uses of C. ternatea and may be recommended for use in the treatment of different types of skin wounds, where taraxerol may be a responsible biomarker.KEY WORDS: Clitoria ternatea, elastase, hyaluronidase, matrix-metalloproteinase-1, taraxerol     

Effect of centchroman coadministration on the pharmacokinetics of metformin in rats

Objectives:

To study the effect of centchroman, a non-steroidal oral contraceptive, coadministration on the pharmacokinetics of metformin in rats.

Materials and Methods:

The pharmacokinetic interaction of metformin was studied in normal Sprague-Dawley female rats with and without centchroman coadministration. Blood samples were analyzed using a validated high-performance liquid chromatography method to generate the pharmacokinetic profile of metformin. The C_max and t_max were directly read from the concentration–time data. Other pharmacokinetic parameters were estimated using non-compartmental analyses.

Results:

Metformin was monitored up to 10 h, and it exhibited a double-peak phenomenon. The C_{max 1}, 2.62 ± 0.32 μg/ml, and C_{max 2,} 2.96 ± 0.65 μg/ml, occurred after 0.75 and 3 h post-dose, respectively. The mean residence time (MRT), AUC_{0-4 h} and volume of distribution (Vd/F) were 4.20 ± 0.30 h, 8.53 ± 1.89 μg.h/ml and 14.24 ± 5.42 L/kg, respectively. Following centchroman coadministration, metformin showed significantly (P < 0.05) higher C_max (C_{max 1,} 3.96 ± 0.55 μg/ml and C_{max 2,} 5.21 ± 0.59 μg/ml), AUC_{0-4 h} (12.28 ± 0.73 μg.h/ml) and Vd/F (18.29 ± 1.19 L/kg), but lower MRT (3.19 ± 0.36 h) than the values obtained after metformin dosing alone. However, AUC_0-t (17.74 ± 5.58 μg.h/ml) and clearance (3.76 ± 0.80 L/h/kg) remained unchanged.

Conclusions:

The results indicate that centchroman coadministration increases the rate but not the extent of absorption of metformin in rats. However, it does not seem to alter the pharmacokinetics of metformin to clinically significant levels.     

Enhanced salt sensitivity following shRNA silencing of neuronal TRPV1 in rat spinal cord

Aim:

To investigate the effects of selective knockdown of TRPV1 channels in the lower thoracic and upper lumbar segments of spinal cord, dorsal root ganglia (DRG) and mesenteric arteries on rat blood pressure responses to high salt intake.

Methods:

TRPV1 short-hairpin RNA (shRNA) was delivered using intrathecal injection (6 μg·kg⁻¹·d⁻¹, for 3 d). Levels of TRPV1 and tyrosine hydroxylase expression were determined by Western blot analysis. Systolic blood pressure and mean arterial pressure (MAP) were examined using tail-cuff and direct arterial measurement, respectively.

Results:

In rats injected with control shRNA, high-salt diet (HS) caused higher systolic blood pressure compared with normal-salt diet (NS) (HS:149±4 mmHg; NS:126±2 mmHg, P<0.05). Intrathecal injection of TRPV1 shRNA significantly increased the systolic blood pressure in both HS rats and NS rats (HS:169±3 mmHg; NS:139±2 mmHg). The increases was greater in HS rats than in NS rats (HS: 13.9%±1.8%; NS: 9.8±0.7, P<0.05). After TRPV1 shRNA treatment, TRPV1 expression in the dorsal horn and DRG of T8-L3 segments and in mesenteric arteries was knocked down to a greater extent in HS rats compared with NS rats. Blockade of α1-adrenoceptors abolished the TRPV1 shRNA-induced pressor effects. In rats injected with TRPV1 shRNA, level of tyrosine hydroxylase in mesenteric arteries was increased to a greater extent in HS rats compared with NS rats.

Conclusion:

Selective knockdown of TRPV1 expression in the lower thoracic and upper lumbar segments of spinal cord, DRG, and mesenteric arteries enhanced the prohypertensive effects of high salt intake, suggesting that TRPV1 channels in these sites protect against increased salt sensitivity, possibly via suppression of sympatho-excitatory responses.     

Magnesium lithospermate B dilates mesenteric arteries by activating BKca currents and contracts arteries by inhibiting Kv currents

Aim:

To examine the involvement of K⁺ channels and endothelium in the vascular effects of magnesium lithospermate B (MLB), a hydrophilic active component of Salviae miltiorrhiza Radix.

Methods:

Isolated rat mesenteric artery rings were employed to investigate the effects of MLB on KCl- or norepinephrine-induced contractions. Conventional whole-cell patch-clamp technique was used to study the effects of MLB on K⁺ currents in single isolated mesenteric artery myocytes.

Results:

MLB produced a concentration-dependent relaxation in mesenteric artery rings precontracted by norepinephrine (1 μmol/L) with an EC₅₀ of 111.3 μmol/L. MLB-induced relaxation was reduced in denuded artery rings with an EC₅₀ of 224.4 μmol/L. MLB caused contractions in KCl-precontracted artery rings in the presence of N-nitro-L-arginine methyl ester (L-NAME) with a maximal value of 130.3%. The vasodilatory effect of MLB was inhibited by tetraethylammonium (TEA) in both intact and denuded artery rings. In single smooth muscle cells, MLB activated BK_Ca currents (EC₅₀ 156.3 μmol/L) but inhibited K_V currents (IC₅₀ 26.1 μmol/L) in a voltage- and concentration-dependent manner.

Conclusion:

MLB dilated arteries by activating BK_Ca channels in smooth muscle cells and increasing NO release from endothelium, but it also contracted arteries precontracted with KCl in the presence of L-NAME.     

Effect of Montelukast on bradykinin-induced contraction of isolated tracheal smooth muscle of guinea pig

Aim:

To explore the effect of montelukast on bradykinin-induced tracheal smooth muscle contraction of isolated guinea pig trachea.

Study Design:

To study the effect of bradykinin in the absence and in the presence of montelukast on the isolated tracheal smooth muscle of a guinea pig pretreated with indomethacin (10^-6M), phentolamine (10^-5M), and propranalol (10^-6M), to eliminate the effect of endogenous prostaglandins and catecholamines. The trachealis smooth muscle activity was recorded through the Isometric Force Displacement Transducer on a Four Channel Oscillograph. A cumulative dose-response relationship was demonstrated by adding successive doses of bradykinin on the tracheal strips, starting with 11 μg to 77 μg of 10^-4 concentration. A similar procedure was repeated in the presence of montelukast 0.5 μg/ml, which, was equal to approximate C_max achieved in vivo with a 10 mg oral dose of montelukast, and in the presence of 1 μg/ml of montelukast.

Statistical Analysis:

Data was expressed as mean ± standard error (SEM), and was analyzed using the SPSS version 15. A P value of less than 0.05 was considered significant.

Results:

Bradykinin produced a dose-dependent, reversible contraction of isolated tracheal smooth muscle. Montelukast significantly reduced the bradykinin-induced tracheal smooth muscle reactivity and shifted the bradykinin curve to the right and downwards, in the presence of both concentrations of montelukast. The mean magnitude of response achieved with 77 μg of bradykinin in the absence of montelukast was 39 mm ± 6.26, in the presence of 0.5 μg/ml of montelukast it was 24.17 mm ± 4.11, and in the presence of 1 μg/ml of montelukast it was 13 mm ± 2.6.

Conclusion:

It is concluded that montelukast significantly inhibits, in a dose-dependent manner, the bradykinin-induced contraction of the guinea pig tracheal smooth muscle, and alludes to an interaction between the bradykinin and leukotriene mediators.     

Effect of anti-tuberculosis therapy on polymorphic drug metabolizing enzyme CYP2C9 using phenytoin as a probe drug

Objectives:

Patients on anti-tuberculosis therapy (ATT) are more prone to drug interactions in the presence of coexisting illnesses which warrant drug therapy. Rifampicin is a strong CYP enzyme inducer while isoniazid is a potent CYP inhibitor. The objective of the study was to find the net effect of one month ATT on CYP2C9 enzyme and to correlate it with respect to the CYP2C9 genetic polymorphisms.

Materials and Methods:

Forty eight newly diagnosed tuberculosis patients were included in the study based on the inclusion-exclusion criteria. Before commencing ATT, they were given a single dose of phenytoin 300 mg as a probe drug for CYP2C9. Blood sample was collected after three hours to carry out CYP2C9 genotyping by PCR-RFLP method. Phenotyping for CYP2C9 enzyme was done by measuring the ratio of phenytoin and its metabolite p-HPPH (para hydroxy phenyl hydantoin) by reverse phase HPLC (high performance liquid chromatography) method before and after one month of ATT.

Results:

In the CYP2C9*1*1 genotype, the mean plasma concentrations of phenytoin before and after one month of ATT were 5.2 ± 0.3 μg/ml and 3.5 ± 0.4 μg/ml respectively, a reduction by 33% showing significant induction (P < 0.001). There was also significant decrease in the metabolic ratio after one month of ATT from 23.2 ± 4.8 to 10.1 ± 1.9 (P < 0.001). The metabolic ratio was also observed to reduce significantly (P < 0.05) when the CYP2C9*1*2, CYP2C9*1*3, and CYP2C9*3*3 data were pooled together.

Conclusion:

The presence of polymorphisms in the CYP2C9 gene does not affect the induction potential of ATT.KEY WORDS: Anti-tuberculosis therapy, CYP2C9, induction, inhibition, pharmacogenetics     

Determination of drug-like properties of a novel antileishmanial compound: In vitro absorption, distribution, metabolism, and excretion studies

In drug discovery research, the compounds should not only to be potent and selective but also must possess acceptable pharmacokinetic properties such as absorption, distribution, metabolism, and excretion (ADME) to increase success rate in clinical studies.

Objective:

Exploration of drug-like properties of 2-(2-methylquinolin-4-ylamino)-N-phenyl acetamide, a potent antileishmanial compound by performing some in vitro ADME experiments along with validation of such studies.

Materials and Methods:

Experimental protocols were established and validated for stability (in PBS pH7.4, simulated gastric and intestinal fluid), solubility, permeability, distribution coefficient (Log D), plasma protein binding and metabolism by rat liver microsomes by using spectrophotometer or HPLC. Methods were considered valid if the results of the standard compounds matched with reported results or within acceptable range or with proper ranking (high-medium-low).

Results:

The compound was found to be stable (>95% remaining) in all stability studies and aqueous solubility was 299.7 ± 6.42 μM. The parallel artificial membrane permeability assay (PAMPA) indicated its medium permeability (Log Pe = −5.53 ± 0.01). The distribution coefficients (Log D) in octanol/PBS and cyclohexane/PBS systems were found to be 0.54 and −1.33, respectively. The plasma protein binding study by the equilibrium dialysis method was observed to be 78.82 ± 0.13% while metabolism by Phase-I enzymes for 1 hour at 37°C revealed that 36.07 ± 4.15% of the compound remained after metabolism.

Conclusion:

The methods were found to be very useful for day-to-day ADME studies. All the studies with the antileishmanial compound ascertained that the compound bears optimum pharmacokinetic properties to be used orally as a potential drug for the treatment of leishmaniasis.     

Interleukin-17A is involved in development of spontaneous pulmonary emphysema caused by Toll-like receptor 4 mutation

Aim:

To explore the pathogenic role of Th17 cells and interleukin-17A (IL-17A)-associated signaling pathways in spontaneous pulmonary emphysema induced by a Toll-like receptor 4 mutant (TLR4^mut).

Methods:

Lungs were obtained from wild-type (WT) or TLR4mut mice that were treated with or without recombinant mouse IL-17A (1 μg·kg⁻¹·d⁻¹, ip) from the age of 3 weeks to 3 months. Pulmonary emphysema was determined using histology, immunochemistry, and biochemical analysis. T cell polarization was determined with flow cytometry, the levels of cytokines were measured using ELISA, and the levels of IL-17A-associated signaling molecules were detected using Western blot.

Results:

Compared to WT mice, 3 month-old TLR4^mut mice were characterized by significantly reduced infiltration of Th17 cells into lungs (2.49%±1.13 % νs 5.26%±1.39%), and significantly reduced expression levels of IL-17A (3.66±0.99 pg/μg νs 10.67±1.65 pg/μg), IL-23 (12.43±1.28 pg/μg νs 28.71±2.57 pg/μg) and IL-6 (51.82±5.45 pg/μg νs 92.73±10.91 pg/μg) in bronchoalveolar lavage fluid. In addition, p38 MAPK phosphorylation and AP-1 expression were decreased to 27%±9% and 51%±8%, respectively, of that in WT mice. Treatment of TLR4^mut mice with IL-17A increased the infiltration of Th17 cells into lungs and expression levels of IL-17A, IL-6, and IL-23 in bronchoalveolar lavage fluid, attenuated MDA and apoptosis, and improved emphysema accompanied with increased phosphorylation of p38 MAPK and expression of AP-1.

Conclusion:

Th17 cells, in particular the cytokine IL-17A, play a crucial role in the pathogenesis of TLR4^mut-induced spontaneous pulmonary emphysema. Both of them are potential targets for therapeutic strategies for pulmonary emphysema.     

Ginsenoside Rbl protects cardiomyocytes against CoCl2-induced apoptosis in neonatal rats by inhibiting mitochondria permeability transition pore opening

Aim:

To investigate whether mitochondria permeability transition pore (mPTP) opening was involved in ginsenoside Rb1 (Gs-Rb1) induced anti-hypoxia effects in neonatal rat cardiomyocytes ex vivo.

Methods:

Cardiomyocytes were randomly divided into 7 groups: control group, hypoxia group (500 μmol/L CoCl₂), Gs-Rb1 200 μmol/L group (CoCl₂ intervention+Gs-Rb1), wortmannin (PI3K inhibitor) 0.5 μmol/L group, wortmannin+Gs-Rb1 group, adenine 9-β-D-arabinofuranoside (Ara A, AMPK inhibitor) 500 μmol/L group, and Ara A and Gs-Rb1 group. Apoptosis rate was determined by using flow cytometry. The opening of the transient mPTP was assessed by using co-loading with calcein AM and CoCl₂ in high conductance mode. Expression of GSK-3β, cytochrome c, caspase-3 and poly (ADP-ribose) polymerase (PARP) was measured by using Western blotting. ΔGSK-3β was defined as the ratio of p-Ser9-GSK-3β to total GSK-3β.

Results:

CoCl₂ significantly stimulated mPTP opening and up-regulated the level of ΔGSK-3β. There was a statistically significant positive correlation between apoptosis rate and mPTP opening, between apoptosis rate and ΔGSK-3β, and between mPTP opening and ΔGSK-3β. Gs-Rb1 significantly inhibited mPTP opening induced by hypoxia (41.3%±2.0%, P<0.001) . Gs-Rb1 caused a 77.3%±3.2% reduction in the expression of GSK-3β protein (P<0.001) and a significant increase of 1.182±0.007–fold (P=0.0001) in p-Ser9-GSK-3β compared with control group. Wortmannin and Ara A significantly inhibited the effect of Gs-Rb1 on mPTP opening and ΔGSK-3β. Gs-Rb1 significantly decreased expression of cytochrome c (66.1%±1.7%, P=0.001), caspase-3 (56.5%±2.7%, P=0.001) and cleaved poly ADP-ribose polymerase (PARP) (57.9%±1.4%, P=0.001).

Conclusion:

Gs-Rb1 exerted anti-hypoxia effect on neonatal rat cardiomyocytes by inhibiting GSK-3β-mediated mPTP opening.     

Antimicrobial clerodane diterpenoids from Microglossa angolensis Oliv. et Hiern

Objective:

To identify the antimicrobial components present in Microglossa angolensis following fractionation of the methylene chloride extract of the aerial part of this plant.

Materials and Methods:

The plant was dried and extracted by percolation with methylene chloride. The dry extract was fractionated and purified by silica gel column chromatography. The isolated compounds were identified by comparison of their Nuclear Magnetic Resonance (NMR) spectral data with those reported in the literature. Antimicrobial activity was assayed by broth macro dilution method.

Results:

The crude extract of M. angolensis displayed significant antifungal and antibacterial activities (MIC = 312.50-1250μg/ml). 6β-(2-methylbut-2(Z)-enoyl)-3α,4α,15,16-bis-epoxy-8β,10βH-ent-cleroda-13(16),14-dien-20,12-olide and spinasterol were the most active compounds (MIC = 1.56-100μg/ml) and the most sensitive microorganisms were Enterococcus faecalis and Candida tropicalis for bacteria and yeasts respectively.

Conclusion:

The isolation of these active antibacterial and antifungal principles supports the use of M. angolensis in traditional medicine for the treatment of gastro-intestinal disorders.     

Comparison of sublingual, vaginal, and oral misoprostol in cervical ripening for first trimester abortion

Objectives:

To compare the effectiveness and tolerability of misoprostol as a cervical ripening agent in first trimester abortion through three different routes of administration before surgical evacuation (SE).

Materials and Methods:

It was a hospital based prospective randomized open labeled parallel study. A total of 150 randomly selected married women were divided in three groups for sublingual (S/L), vaginal and oral 400 μg of misoprostol single dose administration. The drug was administered 3-4 h before SE in the S/L and vaginal groups and 12 h before the procedure in the oral group. Efficacy was assessed on the basis of time taken for ripening, dilatation achieved, duration of the procedure, intra-operative blood loss, and pain score. The tolerability was noted on the basis of side effects.

Results:

The mean time taken for cervical ripening was less in sublingual administration (3.7±1.2 hr) as compared to the vaginal and oral routes. The S/L group had significant cervical dilatation (P<0.001) and the duration of SE was less as compared to the vaginal and oral routes. However, the mean intraoperative blood loss was more in sublingual as compared to the vaginal and oral groups. The intra-operative pain score of the S/L group was significantly lower (1.9±1.1, P<0.05) as compared to the vaginal (2.6±1.7) or oral route (3.3±1.7). Loose motions and nausea/vomiting were more with the S/L and oral routes while blood loss was more in the vaginal route.

Conclusion:

Administration of misoprostol by the sublingual route is better than the oral and vaginal routes for cervical ripening.     

Betulinic acid inhibits superoxide anion-mediated impairment of endothelium-dependent relaxation in rat aortas

Objectives:

To investigate the protective effect of betulinic acid (BA) on endothelium-dependent relaxation (EDR) in rat aortas exposed to pyrogallol-produced superoxide anion and its underlying mechanism.

Materials and Methods:

The thoracic aorta of male Sprague-Dawley rats was isolated to mount in the organ bath system and the effect of BA on acetylcholine (ACh)-induced EDR, nitric oxide (NO) level, reactive oxygen species (ROS) level, nitric oxide synthase (NOS) activity, and superoxide dismutase (SOD) activity of aortic rings exposed to pyrogallol (500 μM) for 15 min were measured.

Results:

BA evoked a concentration-dependent EDR in aortas, and pretreatment with EC₅₀ (2.0 μM) concentration of BA markedly enhanced ACh-induced EDR of aortas exposed to pyrogallol-produced superoxide anion (E_max rose from 23.91 ± 5.41% to 42.45 ± 9.99%), which was markedly reversed by both N^w -nitro-L-arginine methyl ester hydrochloride (L-NAME) and methylene blue, but not by indomethacin. Moreover, BA significantly inhibited the increase of ROS level, as well as the decrease of NO level, the endothelial NOS (eNOS) activity, and the SOD activity in aortas induced by pyrogallol-derived superoxide anion.

Conclusion:

These results indicate that BA reduces the impairment of EDR in rat aortas exposed to exogenous superoxide anion, which may closely relate to the reduction of oxidative stress and activation of eNOS–NO pathway.KEY WORDS: Betulinic acid, endothelium-dependent relaxation, nitric oxide oxidative stress, rat thoracic aorta     

The effect of inhaled corticosteroids on hypothalamic-pituitary-adrenal axis

Objectives:

The aim of this study was to compare systemic effects of high-dose fluticasone propionate (FP) and beclomethasone dipropionate (BDP) via pressurized metered dose inhaler on adrenal and pulmonary function tests.

Materials and Methods:

A total of 66 patients with newly diagnosed moderate persistent asthma without previous use of asthma medications participated in this single blind, randomized, parallel design study. FP or BDP increased to 1 500 μg/d in 62 patients who had not received oral or IV corticosteroids in the previous six months. Possible effects of BDP and FP on adrenal function were evaluated by free cortisol level at baseline and after Synacthen test (250 μg). Fasting plasma glucose and pulmonary function tests were also assessed. Similar tests were repeated 3 weeks after increasing dose of inhaled corticosteroids to 1 500 μg/d.

Results:

No statistically significant suppression was found in geometric means of cortisol level post treatment in both groups. After treatment in FP group, mean forced expiratory volume in one second (FEV1) and mean forced vital capacity (FVC) values improved by 0.17 l (5.66% ± 13.91, P=0.031) and 0.18 l (5.09% ± 10.29, P=0.010), respectively. Although FEV1 and FVC improved in BDP group but was not statistically significant. Oral candidiasis and hoarseness were observed in 6.5% patients receiving BDP, but hoarseness was found in 3.2% patients in FP group (P=0.288).

Conclusions:

The results indicate that safety profiles of high doses of BDP and FP with respect to adrenal function are similar, but FP is more efficacious than that of BDP in improving pulmonary function test.KEY WORDS: Adrenal cortex function tests, adrenal insufficiency, asthma, beclomethasone dipropionate, fluticasone     

Improvement in endothelial dysfunction in patients with systemic lupus erythematosus with N-acetylcysteine and atorvastatin

Objective:

To study the effects of N-acetylcysteine (NAC) and atorvastatin on endothelial dysfunction in patients with systemic lupus erythematosus (SLE).

Materials and Methods:

Thirty-two SLE patients and age, sex-matched 10 healthy control subjects were studied. The patients were between 17 and 65 years of age and positive for diagnostic tests, such as antinuclear antibodies (ANA). Photoplethysmogram (PPG) detects the changes in the amount of light absorbed by hemoglobin, which reflects changes in the blood volume. Pulse wave analysis was performed at rest, 30 s, 90 s after shear stress, and 10 min after 300 μm of salbutamol inhalation.

Results:

Stiffness index (SI) of patients before the treatment was 8.46±2.78 cm/s and of controls was 6.07±1.4 cm/s (P = 0.002) and that of reflection index (RI) was 73±13 for patients and 65±7 for controls (P = 0.001). The percentage change in RI after salbutamol inhalation for controls and patients were -16±6 and -7±4 (P = 0.001), respectively, indicating the presence of endothelial dysfunction. The percentage decrease in RI after salbutamol inhalation was from -2.36±0.76 to ?7.92±1.46 in patients treated with N-acetylcysteine (NAC, P = 0.007). The percentage decrease in RI after salbutamol inhalation was from ?6.36΁1.21 to -9.92±1.21 in patients treated with atorvastatin (P = 0.05). This indicated the improvement in endothelial function. There was decrease in C-reactive protein (CRP) from 1.03±0.72 mg/dL to 0.52±0.22 mg/dL and that of malondialdehyde (MDA) from 11.20±4.07 nmol/mL to 8.81±2.79 nmol/mL with N-acetylcysteine treatment (P < 0.05). The CRP was decreased from 1.11±0.92 mg/dL to 0.440.16 mg/dL (P = 0.05) and that of MDA was decreased from 9.37±3.29 nmol/mL to 8.51±3.27 nmol/mL after treatment with atorvastatin. It showed improvement in oxidative stress with these treatments.

Conclusion:

The presence of arterial stiffness indicated endothelial dysfunction. There was reduction in RI and SI with treatment of N-acetylcysteine and atorvastatin suggesting improvement in endothelial dysfunction. There was decrease in CRP (a marker of inflammation) and MDA after treatment with N-acetylcysteine suggesting improvement in endothelial dysfunction. There was reduction in CRP after treatment with atorvastatin, suggesting improvement in endothelial function. Improvement in endothelial dysfunction is associated with decreased incidence of cardiovascular and cerebrovascular accidents.     

Cytotoxic effect of trans-cinnamaldehyde on human leukemia K562 cells

Aim:

To investigate the effects of trans-cinnamaldehyde (TCA) on the human leukemia K562 cell line and the cytotoxicity of cytokine-induced killer (CIK) cells against K562 cells.

Methods:

Apoptosis, Fas expression, and mitochondrial transmembrane potential in K652 cells were analyzed using flow cytometry. K562 cells were labeled with CFSE. The cytotoxic effect of expanded CIK cells on CFSE-labeled K562 cells was determined by FACS flow cytometry.

Results:

Treatment with TCA 180 μmol/L for 9 h induced apoptosis in 8.9%±1.23% of K562 cells. Treatment with 120 or 180 μmol/L TCA for 24 h significantly increased the apoptotic cells to 18.63%±1.42 % and 38.98%±2.74%, respectively. TCA significantly upregulates Fas expression and decreases mitochondrial transmembrane potential in K562 cells. TCA treatment at 120 and 180 μmol/L for 9 h enhanced the percentage of lysis of K562 cells by expanded CIK cells from 34.84%±2.13% to 48.21%±2.22 % and 64.81%±3.22% at the E:F ratio of 25:1 and from 49.26%±3.22% to 57.81%±5.13% and 73.36%±5.98% at E:F ratio of 50:1.

Conclusion:

TCA exerts cytotoxic effects on human leukemia K562 cells by inducing apoptosis and synergizing the cytotoxicity of CIK cells against K562 cells. These properties of TCA are beneficial to the treatment of leukemia, even in the patients who have received hematopoietic stem cells transplantation (HSCT).     

In vitro antioxidant activity of pet ether extract of black pepper

Objective:

To investigate the in vitro antioxidant activity of different fractions (R1, R2 and R3) obtained from pet ether extract of black pepper fruits (Piper nigrum Linn.)

Materials and Methods:

The fractions R1, R2 and R3 were eluted from pet ether and ethyl acetate in the ratio of 6:4, 5:5 and 4:6, respectively.1,1-Diphenyl-2-picryl-hydrazyl (DPPH) radical, superoxide anion radical, nitric oxide radical, and hydroxyl radical scavenging assays were carried out to evaluate the antioxidant potential of the extract.

Results:

The free radical scavenging activity of the different fractions of pet ether extract of P. nigrum (PEPN) increased in a concentration dependent manner. The R3 and R2 fraction of PEPN in 500 µg/ml inhibited the peroxidation of a linoleic acid emulsion by 60.48±3.33% and 58.89±2.51%, respectively. In DPPH free radical scavenging assay, the activity of R3 and R2 were found to be almost similar. The R3 (100µg/ml) fraction of PEPN inhibited 55.68±4.48% nitric oxide radicals generated from sodium nitroprusside, whereas curcumin in the same concentration inhibited 84.27±4.12%. Moreover, PEPN scavenged the superoxide radical generated by the Xanthine/Xanthine oxidase system. The fraction R2 and R3 in the doses of 1000µg/ml inhibited 61.04±5.11% and 63.56±4.17%, respectively. The hydroxyl radical was generated by Fenton''s reaction. The amounts of total phenolic compounds were determined and 56.98 µg pyrocatechol phenol equivalents were detected in one mg of R3.

Conclusions:

P. nigrum could be considered as a potential source of natural antioxidant.     

Sodium/hydrogen exchange inhibition with cariporide reduces leukocyte adhesion via P-selectin suppression during inflammation

Background and purpose:

The Na⁺/H⁺ exchange (NHE) inhibitor cariporide is known to ameliorate ischaemia/reperfusion (I/R) injury by reduction of cytosolic Ca²⁺ overload. Leukocyte activation and infiltration also mediates I/R injury but whether cariporide reduces I/R injury by affecting leukocyte activation is unknown. We studied the effect of cariporide on thrombin and I/R induced leukocyte activation and infiltration models and examined P-selectin expression as a potential mechanism for any identified effects.

Experimental approach:

An in vivo rat mesenteric microcirculation microscopy model was used with stimulation by thrombin (0.5 μ ml⁻¹) superfusion or ischaemia (by haemorrhagic shock for 60 min) and reperfusion (90 min).

Key results:

Treatment with cariporide (10 mg kg⁻¹ i.v.) significantly reduced leukocyte rolling, adhesion and extravasation after thrombin exposure. Similarly, cariporide reduced leukocyte rolling (54±6.2 to 2.4±1.0 cells min⁻¹, P<0.01), adherence (6.3±1.9 to 1.2±0.4 cells 100 μm⁻¹, P<0.01) and extravasation (9.1±2.1 to 2.4±1.1 cells per 20 × 100 μm perivascular space, P<0.05), following haemorrhagic shock induced systemic ischaemia and reperfusion. The cell adhesion molecule P-selectin showed a profound decrease in endothelial expression following cariporide administration in both thrombin and I/R stimulated groups (35.4±3.2 vs 14.2±4.1% P-selectin positive cells per tissue section, P<0.01).

Conclusions and implications:

The NHE inhibitor cariporide is known to limit reperfusion injury by controlling Ca²⁺ overload but these data are novel evidence for a vasculoprotective effect of NHE inhibition at all levels of leukocyte activation, an effect which is likely to be mediated at least in part by a reduction of P-selectin expression.