Analysis of the cytotoxic, genotoxic, mutagenic, and pro-oxidant effect of synephrine, a component of thermogenic supplements, in human hepatic cells in vitro.

Thermogenic supplements containing synephrine (SN) are widely used to weight loss. SN is a proto-alkaloid naturally found in the bark of immature fruits of Citrus aurantium (bitter orange) that has been added to thermogenic supplements due to its chemical and pharmacological similarity with adrenergic amines, such as ephedrine and amphetamines. Although orally ingested SN is mainly metabolized in the liver, it remains unclear whether it affects the redox status and genetic material of human hepatic cells. The present study aims to examine whether SN affects cell viability, cell cycle, redox balance, genomic stability, and expression of the DNA damage response (DDR)-related genes ATM, ATR, CHEK1, CHECK2, TP53, and SIRT1 in HepG2 cells - used as in vitro hepatocyte model. SN induced overproduction of intracellular reactive oxygen species (ROS) after 6 h of treatment with the three concentrations tested (2, 20 and 200 µM). After 24 h of treatment, SN at 200 µM induced intracellular ROS overproduction and exerted cytostatic effects, while SN at 20 and 200 µM increased the levels of GPx and GSH. SN was not cytotoxic (2-5000 µM), genotoxic, and mutagenic and did not alter the expression of DDR-related genes (2-200 µM), indicating that the fast/specific SN metabolization and upregulation of antioxidant defense components to detoxify intracellular ROS were sufficient to prevent intracellular damage in HepG2 cells. In conclusion, SN showed no cytotoxic, genotoxic, and mutagenic potential at relevant concentrations for thermogenic users in human hepatic cells in vitro, although, it plays pro-oxidative action, and cytostatic effects. Taken together, our results suggest that other investigations about the hazard absence of this thermogenic compound should be performed.

Study to Probe Subsistence of Host-Guest Inclusion Complexes of α and ß-Cyclodextrins with Biologically Potent Drugs for Safety Regulatory Dischargement.

Host-guest interaction of two significant drugs, phenylephrine hydrochloride and synephrine with α and ß-cyclodextrins were studied systematically. Initially two simple but reliable physicochemical techniques namely conductance and surface tension were employed to find out saturation concentration for the inclusion and its stoichiometry. The obtained 1:1 stoichiometry was further confirmed by two spectrometric methods, UV-Vis study and spectrofluorimetry. Significant shifts in IR stretching frequency also support the inclusion process. Relative stabilities of the inclusion complexes were established by the association constants obtained from UV-Vis spectroscopic measurements, program based mathematical calculation of conductivity data. Calculations of the thermodynamic parameters dictates thermodynamic feasibility of the inclusion process. Spectrofluorometric measurement scaffolds the UV-Vis spectroscopic measurement validating stability of the ICs once again. Mass spectroscopic measurement gives the molecular ion peaks corresponding to the inclusion complex of 1:1 molar ratio of host and guest molecules. The mechanism of inclusion was drawn by 1H-NMR and 2D ROESY spectroscopic analysis. Surface texture of the inclusion complexes was studied by SEM. Finally, the cytotoxic activities of the inclusion complexes were analyzed and found, Cell viability also balances for non-toxic behavior of the ICs. Moreover, all the studies reveal the formation of inclusion complexes of two ephedra free, alternatively emerging drugs (after their banned product having ephedra) SNP, PEH with α and ß-CD which enriches the drug delivery system with their regulatory release without any chemical modification.

Citrus aurantium (bitter orange) extract: Safety assessment by acute and 14-day oral toxicity studies in rats and the Ames Test for mutagenicity.

The primary active constituent in bitter orange extract (BOE) is p-synephrine. This study assessed the safety of a BOE standardized to 50% p-synephrine following short-term exposure to rats and by the Ames Test. Following 5000 mg/kg of the extract orally to female rats all animals survived. Administration at 2000 mg/kg to female rats for four days yielded no signs of toxicity. Five male and five female rats were administered the BOE at 0, 250, 500, 1000 and 2000 mg/kg/day for 14 days. No significant effects were observed at any dose with respect to body weights, food intake, absolute and relative organ weights, hematology, clinical chemistry, and pathology. Two male rats died after 2000 mg/kg with gastrointestinal impaction at necropsy. During week two of 1000 mg/kg and 2000 mg/kg/day, rats exhibited transient signs of repetitive burrowing of heads in the bedding material (hypoactivity) for about 15 and 45 min, respectively. The no-observed-effect-level (NOEL) was 500 mg/kg/day. The mutagenic potential was assessed at and up to the limit dose of 5000 µg/plate in a Salmonella typhimurium reverse mutation (Ames) test, performed in duplicate as a pre-incubation assay in the presence and absence of metabolic activation (S9). The BOE did not induce an increase in the frequency of revertant colonies at any dose in the five tester strains, and was therefore non-mutagenic.

Gender differences in biochemical markers and oxidative stress of rats after 28 days oral exposure to a mixture used for weight loss containing p-synephrine, ephedrine, salicin, and caffeine

ABSTRACT The association of p-synephrine, ephedrine, salicin, and caffeine in dietary supplements and weight loss products is very common worldwide, even though ephedrine has been prohibited in many countries. The aim of this study was to evaluate a 28-day oral exposure toxicity profile of p-synephrine, ephedrine, salicin, and caffeine mixture (10:4:6:80 w/w respectively) in male and female Wistar rats. Body weight and signs of toxicity, morbidity, and mortality were observed daily. After 28 days, animals were euthanized and blood collected for hematological, biochemical, and oxidative stress evaluation. No clinical signs of toxicity, significant weight loss or deaths occurred, nor were there any significant alterations in hematological parameters. Biochemical and oxidative stress biomarkers showed lipid peroxidation, and hepatic and renal damage (p < 0.05; ANOVA/Bonferroni) in male rats (100 and 150 mg/kg) and a reduction (p < 0.05; ANOVA/Bonferroni) in glutathione (GSH) levels in all male groups. Female groups displayed no indications of oxidative stress or biochemical alterations. The different toxicity profile displayed by male and female rats suggests a hormonal influence on mixture effects. Results demonstrated that the tested mixture can alter oxidative status and promote renal and hepatic damages.

RESUMO A associação de p-sinefrina, efedrina, salicina, e cafeína em suplementos alimentares e produtos para perda de peso é muito utilizada em todo o mundo, embora a efedrina tenha sido proibida em muitos países. O objetivo deste estudo foi avaliar o perfil de toxicidade à exposição oral de 28 dias à associação de p-sinefrina, efedrina, salicina e cafeína (na proporção de 10:4:6:80 m/m respectivamente) em ratos Wistar machos e fêmeas. Diariamente, os animais foram observados quanto ao peso corporal, sinais de toxicidade, morbidade e mortalidade. Após 28 dias, os animais foram sacrificados e o sangue coletado para avaliações hematológicas, bioquímicas e de estresse oxidativo. Não se observaram sinais clínicos de toxicidade, tampouco perda significativa de peso, mortes, ou quaisquer alterações significativas nos parâmetros hematológicos. Biomarcadores do estresse oxidativo e bioquímicos mostraram peroxidação lipídica, danos renais e hepáticos (p < 0,05; ANOVA/Bonferroni) em ratos machos (100 e 150 mg/kg) e a redução (p < 0,05; ANOVA/Bonferroni) nos níveis de glutationa reduzida (GSH) em todos os grupos de machos tratados. Nas fêmeas, não houve indícios de estresse oxidativo, nem alterações bioquímicas. O diferente perfil de toxicidade entre os gêneros sugere influência hormonal nos efeitos de mistura administrada. A associação testada pode alterar o estado oxidativo e promover danos renais e hepáticos.

Cardiovascular toxicity of Citrus aurantium in exercised rats.

When safety concerns forced the removal of ephedra from the market, other botanicals, including Citrus aurantium or bitter orange (BO) were used as replacements. A major component of the BO extract is synephrine, a chemical that is structurally similar to ephedrine. Because ephedrine has cardiovascular effects that may be exacerbated during physical exercise, the purpose of this study was to determine whether extracts containing synephrine produced adverse effects on the cardiovascular system in exercising rats. Sprague-Dawley rats were dosed daily by gavage for 28 days with 10 or 50 mg of synephrine/kg body weight from one of two different extracts; caffeine was added to some doses. The rats ran on a treadmill for 30 min/day, 3 days/week. Heart rate, blood pressure, body temperature, and QT interval were monitored. Both doses of both extracts significantly increased systolic and diastolic blood pressure for up to 8 h after dosing. Effects on heart rate and body temperature appeared to be due primarily to the effects of caffeine. These data suggest that the combination of synephrine, caffeine, and exercise can have significant effects on blood pressure and do not appear to be effective in decreasing food consumption or body weight.

Physiological effects following administration of Citrus aurantium for 28 days in rats.

BACKGROUND: Since ephedra-containing dietary supplements were banned from the US market, manufacturers changed their formulations by eliminating ephedra and replacing with other botanicals, including Citrus aurantium, or bitter orange. Bitter orange contains, among other compounds, synephrine, a chemical that is chemically similar to ephedrine. Since ephedrine may have cardiovascular effects, the goal of this study was to investigate the cardiovascular effects of various doses of bitter orange extract and pure synephrine in rats. METHOD: Female Sprague-Dawley rats were dosed daily by gavage for 28 days with synephrine from two different extracts. One extract contained 6% synephrine, and the other extract contained 95% synephrine. Doses were 10 or 50mg synephrine/kg body weight from each extract. Additionally, caffeine was added to these doses, since many dietary supplements also contain caffeine. Telemetry was utilized to monitor heart rate, blood pressure, body temperature and QT interval in all rats. RESULTS AND CONCLUSION: Synephrine, either as the bitter orange extract or as pure synephrine, increased heart rate and blood pressure. Animals treated with 95% synephrine showed minimal effects on heart rate and blood pressure; more significant effects were observed with the bitter orange extract suggesting that other components in the botanical can alter these physiological parameters. The increases in heart rate and blood pressure were more pronounced when caffeine was added. None of the treatments affected uncorrected QT interval in the absence of caffeine.

Toxicological effects of a mixture used in weight loss products: p-synephrine associated with ephedrine, salicin, and caffeine.

p-Synephrine is an adrenergic amine found in Citrus aurantium L. fruits and has been used for weight loss in dietary supplements. There are commercial products containing this substance associated to caffeine, salicin, and ephedrine. The aim of this study was to evaluate the acute toxicity of this mixture in mice of both sexes. The significative results observed after acute oral administration to male and female mice of 300, 350, and 400 mg/kg total of p-synephrine, ephedrine, salicin, plus caffeine in a 10:4:6:80 w/w ratio included a reduction in locomotor activity and ptosis in all treated groups for both sexes. Seizures were also observed in male (400 mg/kg) and female groups (350 and 400 mg/kg). Gasping and tearing were observed in males. Salivation (400 mg/kg), agitation (350 and 400 mg/kg), and piloerection (all treated groups) were significantly observed only in females. Deaths occurred in males at 350 and 400 mg/kg treated groups and the necropsy showed cardiopulmonary hemorrhage. A reduction in locomotor activity was confirmed through the spontaneous locomotor activity test, in which the number of crossings considerably decreased (P < .01) in all treated groups. The rotarod test showed a decrease in motor coordination at 400 mg/kg. Body temperature decreased significantly (P < .01) in all treated groups compared to controls. The results suggested clear signs of toxicity of p-synephrine, ephedrine, salicin, and caffeine association; this toxicity augments the attentiveness on commercial products containing this mixture, given the expressive number of adverse events related to its utilization.

Developmental toxicity of Citrus aurantium in rats.

BACKGROUND: Ephedra was commonly used in herbal products marketed for weight loss until safety concerns forced its removal from products. Even before the ban, manufacturers had begun to replace ephedra with other compounds, including Citrus aurantium, or bitter orange. The major component in the bitter orange extract is synephrine which is chemically similar to ephedrine. The purpose of this study was to determine if relatively pure synephrine or synephrine present as a constituent of a bitter orange extract produced developmental toxicity in rats. METHOD: Sprague-Dawley rats were dosed daily by gavage with one of several different doses of synephrine from one of two different extracts. Caffeine was added to some doses. Animals were sacrificed on GD 21, and fetuses were examined for the presence of various developmental toxic endpoints. RESULTS AND CONCLUSION: At doses up to 100 mg synephrine/kg body weight, there were no adverse effects on embryolethality, fetal weight, or incidences of gross, visceral, or skeletal abnormalities. There was a decrease in maternal weight at 50 mg synephrine/kg body weight when given as the 6% synephrine extract with 25 mg caffeine/kg body weight; there was also a decrease in maternal weight in the caffeine only group. This decrease in body weight may have been due to decreased food consumption which was also observed in these two groups. Overall, doses of up to 100 mg synephrine/kg body weight did not produce developmental toxicity in Sprague-Dawley rats.

Synephrine: from trace concentrations to massive consumption in weight-loss.

Synephrine is cited as 'the active component' of plants and dietary supplements used in weight loss. It became one of the most popular stimulants present in weight-loss products after the US Food and Drug Administration had interdicted the use of ephedrine-containing dietary supplements. Synephrine is also a trace amine that can be found in vertebrates and invertebrates. Synephrine acts on several adrenergic and serotonergic receptors and its activity on trace-amine-associated receptors has long been discussed. Synephrine exists in three different positional isomers; however, only p- and m-synephrine have been described in weight-loss products. The alleged effectiveness of synephrine-containing supplements is attributed to the thermogenic effects arising from synephrine's adrenergic stimulation. The growing use of synephrine has raised concerns since it has been accompanied by reports of adverse effects. Cardiac adverse events, including hypertension, tachyarrhythmia, variant angina, cardiac arrest, QT prolongation, ventricular fibrillation, myocardial infarction, and sudden death, have been the most common adverse effects associated with synephrine intake. The mechanisms involved in synephrine-induced cardiotoxicity are still unknown since studies related to its safety are scarce. This review will address general aspects concerning the pharmacology of synephrine, but will focus on the efficacy and toxicity aspects related to the use of synephrine in weight-loss.

Structural isomerization of synephrine influences its uptake and ensuing glutathione depletion in rat-isolated cardiomyocytes.

Synephrine is a natural compound, frequently added to ephedra-free dietary supplements for weight-loss, due to its effects as a nonspecific adrenergic agonist. Though only p-synephrine has been documented in plants, the presence of m-synephrine has also been reported in weight-loss products. The use of synephrine in dietary supplements was accompanied by reports of adverse effects, especially at the cardiovascular level. It is well known that the imbalance in cardiac glutathione levels can increase the risk of cardiomyopathy. The present work aimed to study the role of organic cation-mediated transport of m- and p-synephrine and the possibility that p- and m-synephrine induce intracellular changes in glutathione levels in calcium-tolerant freshly isolated cardiomyocytes from adult rat. After a 3 h incubation with 1 mM p- or m-synephrine, the intracellular content of synephrine was measured by gas chromatography/ion trap-mass spectrometry (GC/IT-MS); cell viability and intracellular glutathione levels were also determined. To evaluate the potential protective effects of antioxidants against the adverse effects elicited by m-synephrine, cells were pre-incubated for 30 min with Tiron (100 µM) or N-acetyl-cysteine (NAC) (1 mM). To assess the influence of α(1)-adrenoceptors activation in glutathione depletion, a study with prazosin (100 nM) was also performed. The results obtained provide evidence that organic cation transporters OCT3 and OCT1 play a major role in m- and p-synephrine-mediated transport into the cardiomyocytes. The importance of these transporters seems similar for both isomers, although p-synephrine enters more into the cardiomyocytes. Furthermore, only m-synephrine induced intracellular total glutathione (GSHt) and reduced glutathione (GSH) depletion. NAC and Tiron were able to counteract the m-synephrine-induced GSH and GSHt decrease. On the other hand, the incubation with prazosin was not able to change m-synephrine-induced glutathione depletion showing that this effect is independent of α(1)-adrenoceptor stimulation. In conclusion, both positional isomers require OCT3 and OCT1-mediated transport to enter into the cardiomyocytes; however, the hydroxyl group in the p-position favours the OCT-mediated transport into cardiomyocytes. Furthermore, the structural isomerization of synephrine influences its toxicological profile since only m-synephrine caused GSH depletion.

Subchronic toxicity of Citrus aurantium L. (Rutaceae) extract and p-synephrine in mice.

Extracts of Citrus aurantium L. (Rutaceae) unripe fruits have gained popularity for the treatment of obesity. Due to the wide use of C. aurantium/p-synephrine-containing products, this research was undertaken to evaluate its subchronic toxicity in mice and their actions in oxidative stress biomarkers. Groups of 9-10 mice received for 28 consecutive days a commercial C. aurantium dried extract (containing 7.5% p-synephrine) 400, 2000 or 4000 mg/kg and p-synephrine 30 or 300 mg/kg by oral gavage. There was a reduction in body weight gain of animals treated with both doses of p-synephrine. Organs relative weight, biochemical and hematological parameters were not altered in all treated mice. There was an increase in reduced glutathione (GSH) concentration in groups treated with C. aurantium 4000 mg/kg and p-synephrine 30 and 300 mg/kg. In glutathione peroxidase (GPx), there were an inhibition of the activity in C. aurantium 400 and 2000 mg/kg and p-synephrine 30 and 300 mg/kg treated animals, respectively, and was no alteration in malondialdehyde (MDA) levels. Thus, the results indicate a low subchronic toxicity of the tested materials in mice and a possible alteration in the oxidative metabolism. However, further tests are required to better elucidate the effects of these compounds in the antioxidant system.

Screening for in vivo (anti)estrogenic activity of ephedrine and p-synephrine and their natural sources Ephedra sinica Stapf. (Ephedraceae) and Citrus aurantium L. (Rutaceae) in rats.

Formulations containing Ephedra sinica Stapf. (Ephedraceae) and Citrus aurantium L. (Rutaceae) are consumed worldwide for body weight control. Considering the related adverse effects and the risk potential, the aim of this study is to evaluate the effects of the thermogenic compounds ephedrine, p-sinephrine, E. sinica and C. aurantium in the female reproductive system through the uterotrophic assay in immature female rats. The animals (n = 6-7) received E. sinica 85.5 and 855.0 mg/kg/day, C. aurantium 25.0 and 50.0 mg/kg/day, ephedrine 5.0 mg/kg/day and p-synephrine 50.0 mg/kg/day for three consecutive days by oral gavage. For detection of antiestrogenicity, tamoxifen 20.0 mg/kg/day, E. sinica 855.0 mg/kg/day, C. aurantium 50.0 mg/kg/day, ephedrine 5.0 mg/kg/day and p-synephrine 50.0 mg/kg/day were administered to estrogen-treated females. Macroscopical alterations were evaluated in liver, kidneys, adrenals and uterus. All analyzed substances showed an antiestrogenic potential, but only ephedrine at 0.5 mg/kg/day presented a significative antiestrogenic effect (P < 0.01). Adrenals relative mass were reduced (P < 0.01) in all tested compounds when compared to the control, which seems to be related to the alfa-1-adrenoceptor agonist activity, which promote a vasoconstriction and reduction of the liquid in the organ. The endocrine system is highly complex and there are a number of ways in which a chemical may interfere with it, other in vivo and in vitro assays are being necessary to support this mechanism of action.

Concentrations of p-synephrine in fruits and leaves of Citrus species (Rutaceae) and the acute toxicity testing of Citrus aurantium extract and p-synephrine.

Dietary supplements containing bitter orange unripe fruit extract/p-synephrine are consumed worldwide for lose weight. This study were conducted to determine the concentration of p-synephrine in unripe fruits and leaves from Citrus aurantium Lin, C. sinensis Osbeck, C. deliciosa Ten, C. limon Burm and C. limonia Osbeck, collected in Southern Brazil, and to evaluate the acute toxicity of C. aurantium extract and p-synephrine. A high performance liquid chromatographic method with diode array detector (HPLC-DAD) was optimized and validated for determination of p-synephrine. The results indicate that all of analyzed samples present p-synephrine in amounts that range from 0.012% to 0.099% in the unripe fruits and 0.029 to 0.438% in the leaves. Acute oral administration of C. aurantium extracts (2.5% p-synephrine, 300-5,000 mg/kg) in mice produced reduction of locomotor activity, p-synephrine (150-2,000 mg/kg) produced piloerection, gasping, salivation, exophtalmia and reduction in locomotor activity, which was confirmed in spontaneous locomotor activity test. All the effects were reversible and persisted for 3-4h. The toxic effects observed seem to be related with adrenergic stimulation and should alert for possible side effects of p-synephrine and C. aurantium.

Molecular mechanisms of toxicity of important food-borne phytotoxins.

At present, there is an increasing interest for plant ingredients and their use in drugs, for teas, or in food supplements. The present review describes the nature and mechanism of action of the phytochemicals presently receiving increased attention in the field of food toxicology. This relates to compounds including aristolochic acids, pyrrolizidine alkaloids, beta-carotene, coumarin, the alkenylbenzenes safrole, methyleugenol and estragole, ephedrine alkaloids and synephrine, kavalactones, anisatin, St. John's wort ingredients, cyanogenic glycosides, solanine and chaconine, thujone, and glycyrrhizinic acid. It can be concluded that several of these phytotoxins cause concern, because of their bioactivation to reactive alkylating intermediates that are able to react with cellular macromolecules causing cellular toxicity, and, upon their reaction with DNA, genotoxicity resulting in tumors. Another group of the phytotoxins presented is active without the requirement for bioactivation and, in most cases, these compounds appear to act as neurotoxins interacting with one of the neurotransmitter systems. Altogether, the examples presented illustrate that natural does not equal safe and that in modern society adverse health effects, upon either acute or chronic exposure to phytochemicals, can occur as a result of use of plant- or herb-based foods, teas, or other extracts.