Pharmacokinetic Analysis of Free Radicals by in vivo BCM (Blood Circulation Monitoring)-ESR Method

In pharmacokinetic studies, a variety of analytical method including radioisotopic detection and HPLC (high performance liquid chromatography) has been used. In the present investigation, we developed in vivo BCM (Blood Circulation Monitoring)-ESR method, which is a new technique with a conventional X-band ESR spectrometer for observing stable free radicals in the circulating blood of living rats under anaesthesia. Both 5–(PROXYL derivatives) and 6–(TEMPO derivatives) membered nitroxide spin probes with various types of substituent functional group were used. After physicochemical properties of the spin probes such as hyperfine coupling constant (A-value), g-value and partition coefficient as well as chemical stability of the compounds in the fresh blood were obtained, the in vivo BCM-ESR method was performed in normal rats. Several pharmacokinetic parameters such as half-life of the probes, distribution volume, total body clearance and mean residence time were obtained and discussed in terms of their chemical structures. In addition, clearance of a spin probe was related to the urine concentration. The BCM-ESR method was found to be very useful to observe free radicals at the real time. By time-dependent ESR signal decay of spin probes, pharmacokinetic parameters were obtained.     

Spinnokinetic analyses of blood disposition and biliary excretion of nitric oxide (NO)-Fe(II)-N-(dithiocarboxy)sarcosine complex in rats: BCM-ESR and BEM-ESR studies

Nitric oxide (NO) is well known to have a wide variety of biological and physiological functions in animals. On the basis of the fact that Fe(II)-dithiocarbamates react with NO, a Fe(II)-N-(dithiocarboxy)sarcosine complex (Fe(II)-DTCS) was proposed as a trapping agent for endogenous NO. However, quantitative pharmacokinetic investigation for NO-Fe(II)-dithiocarbamate complexes in experimental animals has been quite limited. This paper describes the results on the quantitative pharmacokinetic features of a NO-Fe(II)-N-DTCS in both the blood and bile of rats following intravenous (i.v.) administration of the complex. For this purpose, we applied two in vivo methods, i.e. (1) in vivo blood circulation monitoring-electron spin resonance (BCM-ESR) which previously developed, and (2) in vivo biliary excretion monitoring-electron spin resonance (BEM-ESR). We monitored real-time ESR signals due to nitrosyl-iron species in the circulating blood and bile flow. The ESR signal due to NO-Fe(II)-DTCS was stable in biological systems such as the fresh blood and bile. In in vivo BCM- and BEM-ESR, the pharmacokinetic parameters were calculated on the basis of the two-compartment and hepatobiliary transport models. The studies also revealed that the compound is widely distributed in the peripheral organs and partially excreted into the bile. We named a kinetic method to follow spin concentrations as spinnokinetics and this method will be useful for detecting and quantifying the endogenously generated NO in Fe(II)-DTCS administered animals.     

In vivo L-band ESR and quantitative pharmacokinetic analysis of stable spin probes in rats and mice

Free radical species in animals have been measured by X-band ESR spectrometric method on a block of organs or a portion of homogenized samples. However, a nondestructive in vivo ESR measurement has been realized by using a recently developed L-band ESR spectrometry. With this L-band ESR method, we measured ESR spectra in animals, who received stable nitroxide radicals. L-band ESR spectra were observed at the upper abdomen of mice as well as at the heads of mice and rats at various ages immediately after the intravenous injections of nitroxide radicals such as 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl (4-hydroxy-TEMPO) and 3-carbamoyl-2,2,5,5-tetramethyl-pyrrolidine-1-oxyl (3-carbamoyl-PROXYL), in which ESR measurements of the radicals were performed noninvasively at the real time. On the basis of the observed time-dependent free radical clearance curves, the following important results were obtained: (1) Free radical clearances were able to analyze by the pharmacokinetic method. (2) The radicals at the head of mice, given 4-hydroxy-TEMPO, were determined quantitatively by a new analytical method using L-band ESR for the first time. (3) The elimination of the radical was found to be saturated in mice. (4) The clearance rate constant of 4-hydroxy-TEMPO detected at the head of mice was decreased in dose- and age-dependent manners. While, no age-dependent clearance rate constant of 4-hydroxy-TEMPO was observed at the upper abdomen of mice. (5) Ratios of the amount of the detected radicals to that of the administered radicals were decreased age-dependently, but they were independent of the dose of the radicals, suggesting the age-dependent decrease of distribution capacity ratio of the radical at the head of animals. (6) Clearance rate constants of 4-hydroxy-TEMPO and 3-carbamoyl-PROXYL, that were estimated by X- and L-band ESR for the collected blood of mice and rats, were found to be remarkably smaller than those in whole living animals observed by in vivo L-band ESR method. The results suggest that the clearance of the nitroxide radical is relevant to the alteration of the radical in animals following the change of organ distribution and metabolism. (7) Both the radical and its corresponding hydroxylamine, which is the reduced form of the radical, were detectable by X-band ESR method in the collected urine of mice and rats without and with an oxidizing agent, respectively.

On the basis of the results on L-band ESR spectrometry, the first quantitative pharmacokinetic analysis of stable spin probes in animals is proposed.     

Metallokinetic analysis of disposition of vanadyl complexes as insulin-mimetics in rats using BCM-ESR method

Among vanadium's wide variety of biological functions, its insulin-mimetic effect is the most interesting and important. Recently, the vanadyl ion (+4 oxidation state of vanadium) and its complexes have been shown to normalize the blood glucose levels of streptozotocin-induced diabetic rats (STZ-rats). During our investigations to find more effective and less toxic vanadyl complexes, the vanadyl-methylpicolinate complex (VO-MPA) was found to exhibit higher insulin-mimetic activity and less toxicity than other complexes, as evaluated by both in vitro and in vivo experiments. Electron spin resonance (ESR) is capable of measuring the paramagnetic species in biological samples. We have developed the in vivo blood circulation monitoring-electron spin resonance (BCM-ESR) method to analyze the ESR signals due to stable organic radicals in real time. In the present investigation, we have applied this method to elucidate the relationship between the blood glucose normalizing effect of VO-MPA and the global disposition of paramagnetic vanadyl species. This paper describes the results of vanadyl species in the circulating blood of rats following intravenous administration of vanadyl compounds. ESR spectra due to the presence of vanadyl species were obtained in the circulating blood, and their pharmacokinetic parameters were estimated using compartment models. The results indicate that vanadyl species are distributed considerably to the peripheral tissues, as estimated by BCM-ESR, and eliminated from the body through the urine, as estimated by ESR at 77 K. The exposure of vanadyl species in the blood was found to be enhanced by VO-MPA treatment. Given these results, we concluded that the pharmacokinetic character of vanadyl species is closely related with the structure and antidiabetic activity of the vanadyl compounds.     

Effect of anaesthesia-induced alterations in haemodynamics on in vivo kinetics of nitroxyl probes in electron spin resonance spectroscopy

Although the advent of in vivo electron spin resonance (ESR) spectroscopy has allowed analysis of the redox status of living animals, whether the haemodynamic condition affects the signal decay rate remains unknown. Three kinds of haemodynamic conditions were generated by changing the anaesthetic dosage in mice. Haemodynamics was analysed (n=6 each) and in vivo ESR was performed to measure the signal decay rates of three nitroxyl spin probes (carbamoyl-, carboxy- and methoxycarbonyl-PROXYL) at the chest and head regions (n=6 for each condition and probe). Haemodynamic analysis revealed negative inotropic and chronotropic effects on the cardiovascular system depending on the depth of anaesthesia. Although signal decay rates differed among three probes, they were not affected by heart rate alteration. In this study we report the haemodynamics-independent signal decay rate of nitoxyl probes.     

Enhanced intraarticular free radical reactions in adjuvant arthritis rats

One of the reasons of rheumatoid arthritis (RA) development is widely recognized the relation of free radical reactions in tissue injuries. The aim of this study was to evaluate the location where in vivo free radical reactions was enhanced in adjuvant arthritis (AA) model rats using in vivo electron spin resonance (ESR)/nitroxyl spin probe technique. The signal decay after intravenous injection of spin probe was enhanced in AA than that in control and suppressed by the pre-treatment of dexamethasone (DXT). Interestingly, the decay in joint cavity occurred prior to paw swelling of AA and suppressed by a simultaneous injection of free radical scavengers, indicating that the enhancement of free radical reactions in joint cavity of AA rats. This technique would be useful tool to determine the location of the enhanced free radical reactions and evaluate the activity of antioxidant medicine with non-invasive real-time measurement.     

Transfer of nitric oxide from the liver to erythrocytes--an ESR study using nitroglycerin-treated mice

Nitric oxide (NO) formation in the liver and blood of the mouse following intraperitoneal treatment with nitroglycerin (glycerol trinitrate, GTN) was determined using electron spin resonance (ESR) spectroscopy. ESR signals of heme-NO complexes were detected at maximum levels within 5 min in the liver, but increased to a maximum level about 15-30 min later in the blood. GTN is not metabolized to release NO in vitro in the blood of the mouse. The hepatic microsomes which showed the heme-NO complexes ESR signals were incubated with mouse erythrocytes, with the result that a hemoglobin-NO signal was obtained from the erythrocytes. The activities of microsomal cytochrome P-450, the hepatic level of glutathione, and the reduction rate of nitroxide radicals in the in vivo liver, measured using L-band ESR spectroscopy, were temporarily decreased following GTN administration. In conclusion, NO in the liver could be scavenged by circulating erythrocytes, which might minimize NO-induced liver damage.     

Electron spin resonance and spin trapping technique provide direct evidence that edaravone prevents acute ischemia-reperfusion injury of the liver by limiting free radical-mediated tissue damage

A novel free radical scavenger, 3-methyl-1-phenyl-2-pyrazolin-5-one (edaravone), is used for the treatment of acute ischemic stroke and is protective in several animal models of organ injury. We tested whether edaravone is protective against acute liver warm ischemia/reperfusion injury in the rat by acting as a radical scavenger. When edaravone was administered prior to ischemia and at the time of initiation of the reperfusion, liver injury was markedly reduced. Production of oxidants in the liver in this model was assessed in vivo by spin-trapping/electron spin resonance (ESR) spectroscopy. Ischemia/reperfusion caused an increase in free radical adducts rapidly, an effect markedly blocked by edaravone. Furthermore, edaravone treatment blunted ischemia/reperfusion-induced elevation in pro-inflammatory cytokines, infiltration of leukocytes and lipid peroxidation in the liver. These results demonstrate that edaravone is an effective blocker of free radicals in vivo in the liver after ischemia/reperfusion, leading to prevention of organ injury by limiting the deleterious effects of free radicals.     

ESR characterization and metallokinetic analysis of Cr(V) in the blood of rats given carcinogen chromate(VI) compounds.

It has been shown that bio-trace metal elements are related to many diseases and the aging process. For many years, carcinogen hexavalent chromium (VI) has been known to be toxic to animals, but its dynamic toxicological mechanism is not sufficiently elucidated. Bioinorganic chemistry in terms of metallokinetic analysis of beneficial or toxic metal ions and their complexes is an important investigation for understanding their biochemical and physiological roles. We have tried to examine the real-time behavior of paramagnetic metal ions and complexes in animals, in which electron spin resonance (ESR) was capable of measuring paramagnetic species in chemical and biological systems. On the basis of our previous results on stable nitroxide spin probes, we have developed the in vivo blood circulation monitoring-electron spin resonance (BCM-ESR) method to analyze time-dependent ESR signal changes due to paramagnetic metal ions and their complexes in real time. When K2Cr2O7 or Na2Cr2O7 in saline was intravenously administered to rats, two ESR signals due to pentavalent chromium(V) were detectable in the circulating blood of rats. Cr(V) detected in the blood was indicated to be in the CrO(O4) and CrO(S2O2) coordination modes after the study on model complexes. From the changes of ESR signal intensities due to Cr(V) in the blood, the metallokinetic parameters were obtained using the pharmacokinetic analysis and the curve-fitting methods. The obtained results are important for understanding carcinogen chromate in terms of the formation of Cr(V) in animals. In addition, we propose the BCM-ESR method, which is useful to analyze the disposition of paramagnetic metal species in the blood of living animals.     

In Vivo Esr Studies of Antioxidant Activity on Free Radical Reaction in Living Mice Under Oxidative Stress

In vivo antioxidant activity seems to be quite complicate due to multiple interaction with biomaterials and differs from results by in vitro experiments. In vivo estimation of antioxidant activity is performed by measuring TBA reactive substances in blood or hydrocarbon gases in breath, but these systems do not measure free radical reaction but the final products of oxidative reaction. In the present study, we applied in vivo ESR to evaluate antioxidant activity by monitoring the redox reaction of nitroxide radical and clearly found that the nitroxide is very susceptible to oxidative stress in vivo and quite useful to evaluate antioxidant activity non-invasively.     

Non-invasive analysis of reactive oxygen species generated in rats with water immersion restraint-induced gastric lesions using in vivo electron spin resonance spectroscopy

Reactive oxygen species (ROS) are reportedly associated with gastric ulcer. We previously reported the use of an in vivo 300-MHz electron spin resonance (ESR) spectroscopy/nitroxyl probe technique to detect _•OH generation in the stomachs of rats with gastric ulcers induced by NH₄OH. However, this is an acute ulcer model, and the relationship between in vivo ROS generation and lesion formation remains to be clarified. To address this question, the same technique was applied to a sub-acute water immersion restraint (WIR) model. A nitroxyl probe that was less membrane-permeable was orally administered to WIR-treated rats, and the spectra in the gastric region were obtained by in vivo ESR spectroscopy. The signal intensity of the orally administered probe was clearly changed in the WIR group, but no change occurred in the control group. Both enhanced signal decay and neutrophil infiltration into mucosa were observed 2 h after WIR with little formation of any mucosal lesions. The enhanced signal decay was caused by _•OH generation, based on the finding that the decay was suppressed by mannitol, desferrioxamine and catalase. Intravenous treatment with either anti-neutrophil antibody or allopurinol also suppressed the enhanced signal decay, and allopurinol depressed neutrophil infiltration into the mucosa. In rats treated with WIR for 6 h, lesion formation was suppressed by 50% with all antioxidants used in this experiment except anti-neutrophil antibody. These findings suggest that _•OH, which is generated in the stomach via the hypoxanthine/xanthine oxidase system upon neutrophil infiltrated into the mucosa, induces mucosal lesion formation, but that it accounts for only half the cause of lesion formation.     

Estimation of hydroxyl radical generation by salicylate hydroxylation method in multiple organs of mice exposed to whole-body X-ray irradiation

Appropriate experimental conditions for the estimation of hydroxyl radical generation by salicylate hydroxylation were determined for multiple organs of X-irradiated mice in vivo. The in vitro experiments showed that there were significant correlations between the salicylic acid (SA) concentration, the amount of 2,3-dihydroxy benzoic acid (2,3-DHBA) and the X-ray exposure dose, and we obtained two linear-regression equations to calculate the amounts of hydroxyl radicals generated by the X-irradiation. The optimum dosage of SA and the appropriate sampling time for in vivo experiments was determined, and significant increases in the ratio of 2,3-DHBA to SA were detected in several organs of mice after X-irradiation. The hydroxyl radical equivalents of the 2,3-DHBA increases were also calculated. Our results clearly demonstrated the usefulness of the salicylate hydroxylation method in estimating hydroxyl radical generation in multiple organs in vivo.     

蛹虫草MF27高抗氧化活性提取物筛选及保肝作用研究

本研究对一株优质蛹虫草菌株MF27不同提取物进行体外抗氧化活性比较,筛选得到高抗氧化活性提取物,并进一步探究该提取物对CCl₄诱导的小鼠肝损伤的修复作用。以DPPH自由基和羟自由基的清除率为抗氧化评价指标,从菌丝体发酵液、菌丝体水提物/醇提物、以及子实体水提物/醇提物中筛选菌株MF27的高抗氧化活性提取物;以CCl₄致小鼠急性肝损伤为模型,通过检测血清生化指标、肝功指标的变化,来评价该高活性提取物的体内抗氧化保肝效果。体外抗氧化实验结果表明,MF27的不同提取物均具有较好的体外抗氧化活性,但对清除DPPH和OH自由基能力最好的提取物是子实体水提物,其对DPPH自由基的半数有效浓度（EC₅₀）为0.096mg/mL,对羟自由基的半数有效浓度（EC₅₀）为0.196mg/mL,当在1mg/mL 时对DPPH自由基的清除率为94.94%,对羟自由基的清除率为70.17%;体内抗氧化保肝结果显示,给药组（子实体水提物）相比模型组而言,小鼠血清中ALT、AST以及肝脏中MDA水平极显著降低（P<0.01）、SOD水平极显著升高（P<0.01）,表明子实体水提物能有效改善氧化性肝损伤,同时与阳性对照（联苯双酯）对比,给药组在肝脏指数上表现出相当的作用（P>0.05）。本研究表明菌株MF27的最有效抗氧化活性提取物是子实体水提物,它对体内氧化性肝损伤有一定的修复能力,揭示MF27子实体水提物具有成为抗氧化性肝损伤功能产品的潜力。     

Spinnokinetic Analyses of Blood Disposition and Biliary Excretion of Nitric Oxide (NO)-Fe(II)-N-(Dithiocarboxy)sarcosine Complex in Rats: BCM-ESR and BEM-ESR Studies

Nitric oxide (NO) is well known to have a wide variety of biological and physiological functions in animals. On the basis of the fact that Fe(II)-dithiocarbamates react with NO, a Fe(II)-N-(dithiocarboxy)sarcosine complex (Fe(II)-DTCS) was proposed as a trapping agent for endogenous NO. However, quantitative pharmacokinetic investigation for NO-Fe(II)-dithiocarbamate complexes in experimental animals has been quite limited. This paper describes the results on the quantitative pharmacokinetic features of a NO-Fe(II)-N-DTCS in both the blood and bile of rats following intravenous (i.v.) administration of the complex. For this purpose, we applied two in vivo methods, i.e. (1) in vivo blood circulation monitoring-electron spin resonance (BCM-ESR) which previously developed, and (2) in vivo biliary excretion monitoring-electron spin resonance (BEM-ESR). We monitored real-time ESR signals due to nitrosyl-iron species in the circulating blood and bile flow. The ESR signal due to NO-Fe(II)-DTCS was stable in biological systems such as the fresh blood and bile. In in vivo BCM- and BEM-ESR, the pharmacokinetic parameters were calculated on the basis of the two-compartment and hepatobiliary transport models. The studies also revealed that the compound is widely distributed in the peripheral organs and partially excreted into the bile. We named a kinetic method to follow spin concentrations as spinnokinetics and this method will be useful for detecting and quantifying the endogenously generated NO in Fe(II)-DTCS administered animals.     

The detection of hydroxyl radicals in vivo

Several indirect methods have been developed for the detection and quantification of highly reactive oxygen species (hROS), which may exist either as free hydroxyl radicals, bound “crypto” radicals or Fe(IV)-oxo species, in vivo. This review discusses the strengths and weaknesses associated with those most commonly used, which determine the hydroxylation of salicylate or phenylalanine. Chemical as well as biological arguments indicate that neither the hydroxylation of salicylate nor that of phenylalanine can guarantee an accurate hydroxyl radical quantitation in vivo. This is because not all hydroxylated product-species can be used for detection and the ratio of these species strongly depends on the chemical environment and on the reaction time. Furthermore, at least in the case of salicylate, the high concentrations of the chemical trap required (mM) are known to influence biological processes associated with oxidative stress.

Two, newer, alternative methods described, the 4-hydroxy benzoic acid (4-HBA) and the terephthalate (TA) assays, do not have these drawbacks. In each case reaction with hROS leads to only one hydroxylated product. Thus, from a chemical viewpoint, they should provide a better hROS quantitation. Further work is needed to assess any possible biological effects of the required millimolar (4-HBA) and micromolar (TA) concentrations of the chemical traps.     

Effect of Glutathione and N-Acetylcysteine on in Vitro and in VIVO Cardiac Toxicity of Doxorubicin

The effects of two sulfhydryl compounds, glutathione (GSH) and N-acetylcysteine (NAC), on the cardiotoxicity of doxorubicin (DXR) were tested on in vitro and in vivo models. DXR was administered to rats as 4 weekly i.v. doses of 3mg/kg. GSH (1.5 mmoles/kg), given i.v. 10 min before and 1 hr after DXR, was found to prevent the development of the delayed cardiotoxic effects of DXR, as assessed by electrocardiographic and mechanical parameters, as well as by histological examination of left ventricular preparations. In contrast, equimolar oral doses of NAC (1 hr before and 2hrs after DXR) were found to be ineffective. Both GSH and NAC prevented the negative inotropic effect produced by DXR on isolated rat atria. A good correlation exists between the cardioprotective effects of the two agents and their ability to enhance the non-protein sulfhydryl group content of the myocardium. Differences observed in vivo between GSH and NAC might be accounted for by pharmacokinetic factors.     

Structure-dependent metallokinetics of antidiabetic vanadyl-picolinate complexes in rats: studies on solution structure,insulinomimetic activity,and metallokinetics

The insulinomimetic effect of vanadium is the most remarkable and important among its several biological actions. Vanadyl ion (+4 oxidation state of vanadium) and its complexes have been found to normalize the blood glucose levels of both type 1 and 2 diabetic animals. We have developed insulinomimetic vanadyl complexes having different coordination modes, emphasizing the possible usefulness of vanadyl-picolinate [VO(pa)(2)] and its related complexes with the VO(N(2)O(2)) coordination mode. In order to apply these complexes clinically in the future, the relationship between the chemical structure, insulinomimetic action, organ distribution of vanadium, and blood disposition of vanadyl species must be closely investigated. In the present investigation, we studied the blood disposition of the vanadyl-picolinate complexes in healthy rats, and tried to understand comprehensively the relationship between the structures, insulinomimetic activity, and metallokinetic parameters of the complexes, which had been recently prepared and specifically synthesized for the present study, by using an in vivo blood circulation monitoring -- electron spin resonance (BCM-ESR) method for analyzing ESR signals due to paramagnetic metal ions and complexes in the blood in real time. Metallokinetic parameters were estimated based on the blood clearance curves in terms of a two-compartment pharmacokinetic model, and vanadyl species were indicated to be distributed in peripheral tissues and gradually eliminated from the circulating blood, depending on their chemical structures. Vanadyl concentrations in the blood of rats given bis(5-iodopicolinato)oxovanadium(IV) [VO(5ipa)(2)] and bis(3-methylpicolinato)oxovanadium(IV) [VO(3mpa)(2)] with electron-withdrawing and donating groups, respectively, remained significantly higher and longer, due to their slower clearance rates from the blood, than in rats given other complexes, suggesting that the high exposure and long residence of vanadyl species bring about the high normoglyceric effect in diabetic animals. We then examined the relationship between insulinomimetic activity and metallokinetic parameters in the family of VO(pa)(2) for further development of insulinomimetic vanadyl complexes. IC(50), the 50% inhibitory concentration of the complexes on the free fatty acid release from isolated rat adipocytes treated with epinephrine, was found to be sufficiently correlated with metallokinetic parameters such as area under the concentration curve, mean residence time, total clearance, and distribution volume at steady-state. Furthermore, the in vivo antidiabetic activity of the complexes was enhanced with increasing exposure and residence of vanadyl species in the blood of animals. On the basis of these results, we concluded that in vitro insulinomimetic activity, metallokinetic character, and in vivo antidiabetic action of vanadyl-picolinate complexes are closely related to their chemical structures.     

An ESR contrast agent is transported to rat liver through organic anion transporter

Carboxy PROXYL is a useful extracellular paramagnetic contrast reagent in electron spin resonance (ESR) and magnetic resonance imaging (MRI). Active transfer of the probe was investigated using an in situ liver model in rats. Carboxy PROXYL, a nitroxyl spin probe, was perfused into in situ liver perfusion system from Wistar rats. Concentration of nitroxyl form of the spin probe in effluent increased gradually after introducing perfusate with the spin probe and reached a plateau. The disappearance of Carboxy PROXYL from the perfusate was 40%, which could not be explained with its partition coefficient. Administration of non-selective inhibitors of organic anion transporters, p-aminohippuric acid and penicillin G, inhibited competitively and in a dose dependent manner the transfer of Carboxy PROXYL into rat liver in situ, resulting in increases of Carboxy PROXYL in the effluent. The results demonstrate that there is an active transfer system of an ESR contrast reagent into in situ rat liver through organic anion transporters.     

Estrogen-like effect of a Cimicifuga racemosa extract sub-fraction as assessed by in vivo, ex vivo and in vitro assays

Black cohosh (Cimicifuga racemosa) is used in the treatment of painful menstruation and menopausal symptoms. Data about the nature of the active compounds and mechanism(s) of action are still controversial, chiefly with respect to its estrogenic activity.

This work aimed to assess the possible estrogenic activity of a commercial dry hydro-alcoholic extract of C. racemosa and its hydrophilic and lipophilic sub-fractions on in vivo, ex vivo, and in vitro assays.

In a yeast estrogen screen, only the lipophilic sub-fraction was able to activate the human estrogen receptor , with a lower potency but comparable efficacy to that of 17 β-estradiol.

Neither the total extract nor the lipophilic sub-fraction showed an in vivo uterotrophic effect in 21-day-old rats. Uterine tissues obtained ex vivo from C. racemosa treated animals were generally much less sensitive to oxytocin, prostaglandin F_2, and bradykinin than tissues obtained from estradiol valerate treated rats.

The lipophilic sub-fraction, instead, induced a dose-dependent inhibitory activity on the in vitro response to oxytocin, prostaglandin F_2, and bradykinin of uterine horns from naïve 28-day-old rats, with a potency rate close to 1:30 of that of 17 β-estradiol.

Reported results confirm the effectiveness of C. racemosa in menstrual distress and further emphasize the possibility that lipophilic constituents bind to an as yet not identified estrogen receptor, likely inversely involved in inflammation.     

Biological Reduction of Aromatic Nitroso Compounds: Evidence for the Involvement of Superoxide Anions

The in vitro formation of phenylhydronitroxide and 2-methylphenylhydronitroxide free radicals from nitrosobenzene (NB) and 2-nitrosotoluene (NT), respectively, in either red blood cells (RBC) or RBC hemolysates, was confirmed by electron spin resonance spectroscopy (ESR). Free radicals were generated nonenzymatically from reaction of the respective nitroso compounds with a number of biological reducing agents as corroborated by model studies of NB or NT with NAD(P)H. Under aerobic conditions, phenylhydronitroxide and 2-methylphenylhydronitroxide underwent a subsequent one-electron transfer to oxygen, which then resulted in the formation of superoxide anion (O₂^-). The latter product was confirmed by the superoxide dismutase (SOD)-inhibitable reduction of cytochrome c (cyt c). Apparently, oxygen is needed for continuous formation of the hydronitroxide radical derivatives. On the other hand, under anaerobic conditions, no phenylhydronitroxide radical was generated from NB in the presence of NADH, but the formation of phenylhydroxylamine from NB was detected by the absorption spectrometry. These results suggest that oxygen is a preferential electron acceptor for hydronitroxide radical derivatives.