Ethyl acetate fraction of the root of Rubia cordifolia L. inhibits keratinocyte proliferation in vitro and promotes keratinocyte differentiation in vivo: potential application for psoriasis treatment.

Psoriasis is a skin disease associated with hyperproliferation and aberrant differentiation of keratinocytes. Our previous studies have identified the root of Rubia cordifolia L. as a potent antiproliferative and apoptogenic agent in cultured HaCaT cells (IC(50) 1.4 microg/ml). In the present study, ethanolic extract of Radix Rubiae was fractioned sequentially with hexane, ethyl acetate (EA), n-butanol and water. EA fraction was found to possess most potent antiproliferative action on HaCaT cells (IC(50) 0.9 microg/ml). Mechanistic study revealed that EA fraction induced apoptosis on HaCaT cells, as it was capable of inducing apoptotic morphological changes. Annexin V-PI staining assay also demonstrated that EA fraction significantly augmented HaCaT apoptosis. In addition, EA fraction decreased mitochondrial membrane potential in a concentration- and time-dependent manner. The standardized EA fraction was formulated into topical gel and its keratinocyte-modulating action was tested on mouse tail model. EA fraction dose-dependently increased the number and thickness of granular layer and epidermal thickness on mouse tail skin, indicative of the keratinocyte differentiation-inducing activity. Taking the in vitro and in vivo findings together, the present preclinical study confirms that EA fraction is a promising antipsoriatic agent warranting further development for psoriasis treatment.

The skin prick test results of 977 patients suffering from chronic rhinitis in Hong Kong.

OBJECTIVES: To evaluate the prevalence of aetiological allergens identifiable by skin prick test alone in patients suffering from chronic rhinitis in Hong Kong, and also compare the clinical history and symptoms of skin prick test-positive versus skin prick test-negative patients. DESIGN: Prospective study. SETTING: Otorhinolaryngology clinic in Queen Mary Hospital of Hong Kong. PATIENTS: A total of 977 patients suffering from chronic rhinitis were recruited into the study. Skin prick test was performed with a panel of allergens including house dust mites, cockroach, cat, dog, moulds, and pollens. MAIN OUTCOME MEASURES: Skin prick test results and their correlation with symptoms. RESULTS: Of the 977 patients, 651 (67%) had positive skin prick test reactions. The commonest allergen was house dust mite which was positive in 63% of the 977 patients and 95% of those 651 skin prick test-positive patients. The other allergens were in order of cockroach (23%), cat (14%), dog (5%), pollen (4%), and mould (3%). Compared with skin prick test-negative patients, skin prick test-positive patients were more likely to have earlier age of onset of the chronic rhinitis, association with asthma, more severe symptom in the morning, more severe symptoms of itchy nose, sneezing, nasal discharge, itchy eye, and watery eye. CONCLUSIONS: Identifiable aeroallergens could be detected in 67% chronic rhinitis patients by skin prick test alone. House dust mites were the most prevalent causative allergen. There were significant differences of patterns of clinical history and symptoms severity between skin prick test-positive and skin prick test-negative patients.

VITATOPS, the VITAmins TO prevent stroke trial: rationale and design of a randomised trial of B-vitamin therapy in patients with recent transient ischaemic attack or stroke (NCT00097669) (ISRCTN74743444).

BACKGROUND: Epidemiological studies suggest that raised plasma concentrations of total homocysteine (tHcy) may be a common, causal and treatable risk factor for atherothromboembolic ischaemic stroke, dementia and depression. Although tHcy can be lowered effectively with small doses of folic acid, vitamin B(12) and vitamin B(6), it is not known whether lowering tHcy, by means of B vitamin therapy, can prevent stroke and other major atherothromboembolic vascular events. AIM: To determine whether the addition of B-vitamin supplements (folic acid 2 mg, B(6) 25 mg, B(12) 500 microg) to best medical and surgical management will reduce the combined incidence of stroke, myocardial infarction (MI) and vascular death in patients with recent stroke or transient ischaemic attack (TIA) of the brain or eye. DESIGN: A prospective, international, multicentre, randomised, double blind, placebo-controlled clinical trial. SETTING: One hundred and four medical centres in 20 countries on five continents. SUBJECTS: Eight thousand (6600 recruited as of 5 January, 2006) patients with recent (<7 months) stroke (ischaemic or haemorrhagic) or TIA (brain or eye). RANDOMISATION: Randomisation and data collection are performed by means of a central telephone service or secure internet site. INTERVENTION: One tablet daily of either placebo or B vitamins (folic acid 2 mg, B(6) 25 mg, B(12) 500 mug). PRIMARY OUTCOME: The composite of stroke, MI or death from any vascular cause, whichever occurs first. Outcome and serious adverse events are adjudicated blinded to treatment allocation. SECONDARY OUTCOMES: TIA, unstable angina, revascularisation procedures, dementia, depression. STATISTICAL POWER: With 8000 patients followed up for a median of 2 years and an annual incidence of the primary outcome of 8% among patients assigned placebo, the study will have at least 80% power to detect a relative reduction of 15% in the incidence of the primary outcome among patients assigned B vitamins (to 6.8%/year), applying a two-tailed level of significance of 5%. CONCLUSION: VITATOPS aims to recruit and follow-up 8000 patients between 1998 and 2008, and provide a reliable estimate of the safety and effectiveness of folic acid, vitamin B(12), and vitamin B(6) supplementation in reducing recurrent serious vascular events among a wide range of patients with TIA and stroke throughout the world.

Severe immune haemolysis after standard doses of Penicillin.

Summary Penicillin causes immune haemolytic anaemia by the 'drug-adsorption' mechanism and typically occurs after prolonged exposure to large doses of the drug. Withdrawal of the drug is associated with improved red cell survival and gradual cessation of haemolysis. Although this complication is uncommon, it can be potentially serious. An unusual case is described herein. The patient was exposed to a short course (9 days) of standard dose penicillin but suffered acute severe haemolysis about 1 week after cessation of therapy. A high titre anti-penicillin antibody (1 : 512) not cross-reacting with cephalosporins, was demonstrated. The delay in the development of immune haemolysis vis-à-vis penicillin therapy may be due to the patient being immunologically naive to the drug. Penicillin may persist for weeks in circulation, coating red cells and providing continued antigenic stimulation for the development of anti-penicillin antibody.

Characterization of antioxidants present in hawthorn fruits.

Hawthorn fruit extract has been shown to have many health benefits including being cardiovascular protective, hypotensive and hypocholesterolemic. The present study was carried out to characterize further the antioxidants of hawthorn fruit and their effect on the oxidation of human low density lipoprotein (LDL) and alpha-tocopherol. The dry hawthorn fruit was extracted successively with ether, ethyl acetate, butanol and water. The ethyl acetate fraction was only effective in inhibition of Cu(+2)-mediated LDL oxidation. The column chromatographic separation led to isolation of eight pure compounds; namely, ursolic acid, hyperoside, isoquercitrin, epicatechin, chlorogenic acid, quercetin, rutin and protocatechuic acid. All of these phenolic compounds, except ursolic acid, were protective to human LDL from Cu(+2)-mediated LDL oxidation. They were also effective in preventing the peroxy free radical-induced oxidation of alpha-tocopherol in human LDL. The inhibitory effect of these compounds on oxidation of LDL and alpha-tocopherol was dose-dependent at concentrations ranging from 5 to 40 µM. In addition, supplementation of 2% hawthorn fruit powder significantly elevated serum alpha-tocopherol by 18-20% in rats fed a 30% polyunsaturated canola oil diet, as compared with the control. The present results suggest that part of the mechanism for cardiovascular protective effects of hawthorn fruit might also involve the direct protection to human LDL from oxidation or indirect protection via maintaining the concentration of alpha-tocopherol in human LDL.

Inhibitory effects of purified green tea epicatechins on contraction and proliferation of arterial smooth muscle cells.

AIM: The present study was aimed to examine the vasorelaxant and antiproliferative responses to purified green tea epicatechin mixture, (-)epicatechin and (-)epigallocatechin gallate on rat arterial smooth muscle cells. METHODS: Changes in isometric force were measured by Grass force transducer and effects on cell proliferation were evaluated using [3H]thymidine incorporation assay. RESULTS: Epicatechin mixture, (-)epicatechin and (-)epigallocatechin gallate, which we isolated and purified from jasmine green tea, concentration-dependently, reduced the contractile response to phenylephrine in rat isolated aortic rings with (-)epigallocatechin gallate being more effective. These three agents also inhibited [3H]thymidine incorporation into DNA in cultured rat aortic smooth muscle cells in a concentration-dependent manner. CONCLUSION: The purified epicatechin derivatives from jasmine green tea relaxed the isolated rat arteries preconstricted by phenylephrine and inhibited aortic smooth muscle cell proliferation.

Jasmine green tea epicatechins are hypolipidemic in hamsters (Mesocricetus auratus) fed a high fat diet.

These studies were designed to test the hypolipidemic activity of green tea epicatechins (GTE) isolated from jasmine green tea. In Experiment 1, three groups of hamsters were given a semisynthetic diet containing 200 g lard/kg and 1 g cholesterol/kg for 4 wk. The control group received distilled water, and the other two groups received either 15 g/L green tea water extract (GTWE) or 5.0 g/L GTE solution. Both the GTWE and GTE groups had lower concentrations of serum total cholesterol (TC) and triacylglycerols (TG) than the controls (P < 0.05). In Experiment 2, four groups of hamsters received tap water as the drinking fluid, but they were given the same high fat and cholesterol diet supplemented with 0 (control), 1.1, 3.4 or 5.7 g GTE/kg diet. The hypolipidemic effect of jasmine GTE was dose dependent. In Experiment 3, the time-course of changes in serum TC and TG was monitored in hamsters given the high fat diet supplemented with 5.7 g GTE/kg in comparison with that of controls. The hypolipidemic effects of dietary GTE were evident after feeding for 2 wk. Dietary supplementation of GTE did not affect liver fatty acid synthase. However, GTE-supplemented hamsters had higher fecal excretions of total fatty acids, neutral sterols and acidic sterols compared with the control group. In Experiment 4, hamsters were fed nonpurified diet; the control group drank distilled water, and the GTE group drank distilled water containing 5.0 g GTE/L. No differences in activities of 3-hydroxy-3-methyl glutaryl coenzyme A reductase and intestinal acyl CoA:cholesterol acyltransferase were observed. This study suggests that the hypolipidemic activity of GTE is not due to inhibition of synthesis of cholesterol or fatty acid but is most likely mediated by its influence on absorption of dietary fat and cholesterol.

Endothelium-dependent relaxation induced by hawthorn extract in rat mesenteric artery.

The extract prepared from hawthorn (Crataegus fruit) was examined for its relaxant effect in rat isolated mesenteric arteries. Hawthorn extract induced concentration-dependent relaxation of the U46619-precontracted artery with an IC50 of 0.22 +/- 0.02 mg/ml. Removal of the functional endothelium reduced by approximately 85% the maximum relaxant response to hawthorn extract. Pretreatment of the arterial tissues with N(G)-nitro-L-arginine methyl ester (3-10 microM) or methylene blue (3-10 microM) inhibited the relaxation induced by hawthorn extract, while indomethacin (10 microM) had no effect. L-arginine (3 mM) did not affect the relaxation induced by hawthorn extract but partially reversed the effect of 10 microM N(G)-nitro-L-arginine methyl ester. Iberiotoxin (100 nM) slightly but significantly inhibited the relaxant effect of hawthorn extract whilst glibenclamide (3 microM) was ineffective. Glibenclamide at 3 microM reversed the relaxation induced by pinacidil. N(G)-nitro-L-arginine methyl ester and methylene blue markedly inhibited acetylcholine-induced relaxation in endothelium-intact arteries. Hawthorn extract also reduced the contraction induced by phenylephrine (1 microM) or high Ki (60 mM) with respective IC50 values of 0.13 +/- 0.01 mg/ml and 0.11 +/- 0.01 mg/ml. In high K+-contracted arteries, hawthorn extract induced only 55% of relaxation while it caused a complete inhibition of the U46619- or phenylephrine-induced contraction. These results suggest that hawthorn contains active components which cause vasorelaxation in rat isolated mesenteric arteries. Nitric oxide but not other endothelium-derived vasoactive factors was probably involved in the relaxation induced by hawthorn extract.

Opioid-like activity in the cerebrospinal fluid of pain patients treated by electroacupuncture.

Thirteen patients with pain from various causes were treated by electroacupuncture for 30 min. Cerebrospinal fluid (CSF) was obtained before and after treatment. Opioid-like substances in the CSF were fractionated by high pressure liquid chromatography and assayed by competitive receptor binding using a mu-specific radioligand, [D-ala2, MePhe4, gly-ol5]-enkephalin (DAGO). Opioid activity, associated with a fraction, eluted at 18-20% acetonitrile, consistently showed an increase in level after acupuncture. Two other fractions eluted at larger concentrations of acetonitrile also increased significantly after acupuncture; however the increase was not consistently observed in every patient. Measurements of beta-endorphin and dynorphin by radioimmunoassay indicated that 80 and 60% of the patients, respectively, had a higher level of these peptides after acupuncture. The nature of the opioid activity, eluted at 18-20% acetonitrile is unknown; however a small amount of it could be found in various parts of the brain of rat.

Reduction of opioid binding in neuroblastoma x glioma cells grown in medium containing unsaturated fatty acids.

Neuroblastoma x glioma cells NG108-15 were cultured in lipid-free medium supplemented with fatty acids of various chain length and unsaturation. Binding of 3H-labelled [DAla2]-[DLeu5]-enkephalin by membranes of cells grown in saturation fatty acids of different chain length was not significantly different from that of the control On the other hand, a proportional decrease of binding capacity with no change in residual receptor affinity was noticed when cells were cultured in medium containing fatty acids of increasing unsaturation. This decrease was time dependent and reached a maximum at about 48 h. Binding of [3H]dihydromorphine and [3H]naloxone was similarly affected. In contrast, when membranes of cells grown in normal medium were preincubated up to 3 h with unsaturated fatty acid and tested for opioid binding, no significant reduction was observed. Examination of the fatty acid composition of phospholipid from cells grown in linolenate indicated that a significant alteration of the acyl composition has occurred. To evaluate the underlying cause of this type of inhibition, the effect of linolenic acid on cell growth and protein synthesis was examined. When cells were cultured in 100 microM of this fatty acid, both growth and protein synthesis were retarded by 28% and 19%, respectively. Since opiate receptors are proteineous in nature, a reduction of protein synthesis may partially account for the loss of opioid binding activity. On the other hand, an increase of membrane fluidity is known to affect a number of cellular functions, including ligand-receptor recognition. Whether this can offer a satisfactory explanation for our observations remains to be established.

Immunoassayable beta-endorphin level in the plasma and CSF of heroin addicted and normal subjects before and after electroacupuncture.

The present study was undertaken to evaluate if plasma or CSF beta-endorphin level can be induced to rise during the treatment of heroin addiction by electroacupuncture. Based on the examination of 30 addicts, we obtained no evidence indicating an increase of beta-endorphin level in either the plasma or the CSF after 30 min of acupuncture. In spite of this, the majority of the addicts experienced a reduction of withdrawal symptoms during treatment. Since electroacupuncture may only induce a highly localized secretion of beta-endorphin in the brain, our results cannot unequivocally exclude the possibility that this peptide is involved in mediating the action of acupuncture.

Changes in adrenocorticotropic hormone (ACTH) and cortisol levels in drug addicts treated by a new and rapid detoxification procedure using acupuncture and naloxone.

Plasma ACTH, cortisol, and cyclic-AMP levels of eleven heroin addicts were dertermined before and after treatment with a fast detoxification procedure using acupuncture and electrical stimulation (AES) together with the administration of limited doses of naloxone. At the end of the treatment period, the average plasma ACTH, cortisol, and cyclic-AMP level rose 130,83, and 24 percent, respectively. In view of the appearance of mild withdrawal signs during this method of treatment, the observed increases in ACTH and cortisol levels probably reflect the inability of AES to suppress withdrawal symptoms induced by naloxone completely. The mechanism underlying this new method of treatment is discussed in relation to AES's ability to stimulate the secretion of endorphins.

The influence of electro-acupuncture on naloxone-induced morphine withdrawal. II. Elevation of immunoassayable beta-endorphin activity in the brain but not the blood.

The effect of electro-acupuncture in the treatment of naloxone precipitated morphine withdrawal in mice was studied. Thirty minutes of acupuncture was enough to suppress withdrawal behaviors by more than 60%. Concomitant to the decrease of withdrawal behavior, there was a significant increase of brain beta-endorphin level in the acupunctured animals. Plasma beta-endorphin level of the treated and untreated group was not significantly different.

Acupuncture in narcotic withdrawal: a preliminary report on biochemical changes in the blood and urine of heroin addicts.

Eight female heroin-addicted subjects were treated over a 10-day period by acupuncture and electrical stimulation (AES). Their plasma ACTH, cortisol and c-AMP levels were reduced after initial AES treatment. The reduction of c-AMP was significant the most of the three. The suppression of these compounds may be associated with the temporary relief of withdrawl symptoms on and after each AES treatment in the first three days. On the third day, ACTH, cortisol and c-AMP levels did not show significant changes after AES. This was presumably due to the disappearance of withdrawal symptoms and possibly to the stabilizing effect of the treatment. Both the ACTH and c-AMP levels were generally lower at the end of the treatment period. Results of the present investigation suggest that plasma c-AMP may be the best parameter by which to gauge the response of heroin addicts to AES.

Reduction of adrenocorticotropic hormone (ACTH) and cortisol in drug addicts treated by acupuncture and electrical stimulation (AES).

Forty-two heroin addicts and 31 normal persons were examined for the effect of acupuncture and electrical stimulation (AES) on plasma ACTH, cortisol and cyclic-AMP levels. Both ACTH and cortisol levels were reduced significantly in the addicts after treatment whereas no such significant reduction was observed in the normals. Plasma cyclic-AMP level was not affected in either group. Taken together, results from the present study suggest that the mechanism of AES in the treatment of addiction may have a neuroendocrinological basis. This hypothesis is particularly attractive in view of the isolation of opiate-like peptides from the brain.