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

1Laboratory of Analysis and Toxicological Research, Department of Analysis, Faculty of Pharmacy, Federal University of Rio Grande do Sul, Porto Alegre, RS, Brazil, 2REQUIMTE, Laboratory of Toxicology, Department of Biological Sciences, Faculty of Pharmacy, University of Porto, Porto, Portugal, 3Department of Pharmacoscience, Federal University of Heath Science of Porto Alegre, Porto Alegre, RS, Brazil, 4Laboratory of Pharmacology and Toxicology of Natural Products, Department of Pharmacology, Institute of Basic Health Sciences, Federal University of Rio Grande do Sul, Porto Alegre, RS, Brazil


INTRODUCTION
Overweight and obesity are highly prevalent worldwide.Many consumers are seeking strategies for reducing weight to healthier levels, including the use of dietary supplements and weight loss products.These products typically stimulate or enhance weight loss through mechanisms such as reducing hunger or increasing resting metabolism (Dwyer et al., 2005).There are a large number of products being marketed for weight loss, most usually containing a mixture of ingredients including p-synephrine (Citrus aurantium L.), ephedrine (Ma Huang, Ephedra sinica, Ephedra sp.), salicin (Salix sp.) and caffeine (Paullinia cupana, Cola nitida, Cola acuminata, Camellia sinensis), as well as vitamins, minerals, and other herbs (Arbo et al., 2008(Arbo et al., , 2009a(Arbo et al., , 2009b)).
Ephedrine is an adrenergic agonist for both α and β receptors affecting both the cardiovascular and central nervous system (Schaneberg et al., 2003).The use of p-synephrine, a biogenic amine found in Citrus aurantium, became very popular after the banning of ephedrine-containing dietary supplements by the Food and Drug Administration (FDA) due to the occurrence of significant adverse effects, mainly related to the cardiovascular system (Bouchard et al., 2005).It quickly replaced ephedrine in supplements called "ephedra-free" due to their structural similarity (Fugh-Berman, Myers, 2004;Jordan et al., 2004;Grollman, 2005).
However, like ephedrine, p-synephrine is an α and β adrenergic agonist and both are structurally and pharmacologically related to amphetamines (Firenzuoli et al., 2005) and other adrenergic amines such as epinephrine and norepinephrine (Rossato et al., 2011a).For this reason, it is believed that it may also cause adverse effects, especially on the cardiovascular system (Bouchard et al., 2005).On the other hand, Rossato et al. (2011b) observed that only m-synephrine (positional isomeric form of synephrine) induced a potentially toxic effect, while p-synephrine showed negligible cardiotoxicity in an in vitro model.Arbo et al. (2008Arbo et al. ( , 2009aArbo et al. ( , 2009b) ) demonstrated that extracts of both C. aurantium and isolated p-synephrine showed low toxicity in mice, even at high doses.
These facts therefore lead us to believe that reports of toxicity from this amine could be related to its association with other substances, specifically caffeine, salicin and even ephedrine in many products (Schmitt et al., 2012).Although ephedrine has been banned or restricted in several countries, it can be found in some formulations due to adulterations to improve product performance (Schmitt et al., 2012).Caffeine, a stimulant with α-adrenergic-like properties, acts on cardiovascular system (Kalman et al., 2002) and may enhance the effects of sympathomimetics such as ephedrine (Astrup et al., 1992;Haller et al., 2004) and probably synephrine (Schmitt et al., 2012) because their structural similarities (Pellati and Benvenuti, 2007).
Salicin is a phenolic glycoside extracted from Salix sp.(white willow bark), which displays analgesic properties (Zaugg et al., 1997), but there is no scientific support for its presence in weight loss formulations.It is believed to enhance the effects of other substances in a synergistic pathway, extending or increasing the activity of thermogenic ingredients through prolonging ephedrine action time (Schmitt et al., 2012).Acetylsalicylic acid, which belongs to the same salicylate family as salicin, showed increases in the thermogenesis effects of both ephedrine and caffeine in obese women (Horton, Geissler, 1991).
The association of p-synephrine with ephedrine, salicin, and caffeine is found in several marketed products, and it is known that the association of similarly acting substances can increase and/or strengthen their pharmacological actions.Acute toxicity of this association was seen by Schmitt et al. (2012).However, these products are usually consumed over long periods and it is necessary to evaluate the effects after repeated doses.The aim of this study was to evaluate 28 days oral exposure to a mixture of p-synephrine, ephedrine, salicin and caffeine (10:4:6:80 w/w respectively) in male and female rats and their actions on oxidative stress biomarkers.The relative proportions of the substances in the mixture were based on several weight loss products available on the market.However, this does not linearly correspond to products used by humans because proportions and dose indications vary greatly from product to product.

Animals
Experiments were performed after approval from the University Ethics Committee (deliberation number 2007982) and were carried out in accordance with current guidelines for the care of laboratory animals (Goldim, Raymundo, 1997;Olfert et al., 1998).Male and female Wistar rats, weighing 306.3 ± 24.5 g and 196.9 ± 17.6 g respectively, were obtained from Central Animal House of the Institute of Basic Health Sciences, Federal University of Rio Grande do Sul.Animals were housed in 47 × 34 × 18 cm polyethylene cages under standard temperature conditions (22 ± 2 °C), controlled humidity, and a 12 h light/dark cycle.All experimental procedures were performed during the light phase of the cycle.

Repeated dose 28-day oral toxicity study
T h e e x p e r i m e n t a l p r o t o c o l w a s b a s e d o n OECD (Organization for Economic Co-operation and Development) guideline 407 (Repeated Dose 28-day Oral Toxicity Study in Rodents).The experiment was conducted distributing the rodents in groups of 8 male and 8 female rats each.Animals were treated by oral gavage for 28 consecutive days with water (control) or 50, 75, 100, and 150 mg/kg of an aqueous mixture of p-synephrine, ephedrine, salicin and caffeine (proportion of 10:4:6:80 w/w respectively) administered at a constant volume of 10 mL/kg.The substance proportions in the mixture were defined considering several weight loss products available in the market.
Body weight was measured every day and animals were observed daily for signs of toxicity, morbidity, and mortality.At the end of the experiment, animals were euthanized with ketamine/xylazine (50 mg/kg and 10 mg/kg respectively) and necropsied.Selected organs (heart, liver, lung, brain, spleen, kidneys, and adrenal glands) were collected and observed for macroscopic alterations.

Biochemical and hematological analysis
After euthanasia, blood was collected from caudal vein using heparin or EDTA as anticoagulant for biochemical and hematological evaluations.In whole blood with EDTA, hemoglobin and hematocrit were performed according to ICHS (1978) and ICHS (1982) respectively.Part of the blood was centrifuged and heparinized plasma used to evaluated alanine aminotransferase (ALT), aspartate aminotransferase (AST) and creatinine using commercial kits (Labtest Diagnóstica).

Oxidative stress biomarker evaluations
Part of the blood collected with anticoagulant was centrifuged at 1500g for 10 minutes and immediately separated into plasma and erythrocytes.EDTA plasma was used to evaluate lipid peroxidation through an HPLC method with visible detection at 532 nm (Grotto et al., 2007).This method quantifies malondialdehyde (MDA) levels after alkaline hydrolysis and extraction with n-butanol.Erythrocytes were used to determinate reduced glutathione (GSH), which was measured as non-protein thiols, according to Ellman (1959) modified.Aliquots (0.3 mL) of erythrocytes were added to a phosphate buffer 0.3 M (0.85 mL), pH 7.4 and the reaction was read in spectrophotometer at 412 nm after addition of 10 mM 5,5'-dithiobis(2-nitrobenzoic) acid (DTNB) (0.05 mL).The results were expressed as µmol/mL erythrocytes.
The whole blood collected with heparin was used to analyze antioxidant enzyme glutathione peroxidase (GPx) activity using glutathione reductase and NADPH.The method is based on the oxidation of NADPH, which is indicated by a decrease in absorbance at 340 nm (Paglia, Valentine, 1967).

Statistical analysis
Data were expressed as mean ± SEM (standard error of mean).Organ weights were determined and expressed as relative weight and were analyzed by oneway analysis of variance (ANOVA).Body weight gain was evaluated by repeated measures ANOVA.Relative body weight gain was obtained considering initial weight measurement as 100% and all gain was compared to the initial measurement.Differences between groups were evaluated by one-way ANOVA, followed by post hoc Bonferroni, when appropriate.The significance level was set at 5% (p < 0.05).

RESULTS AND DISCUSSION
Daily oral exposure (28 days) of p-synephrine associated to ephedrine, salicin and caffeine in male and female rats did not show signs of toxicity; there were also no deaths of either male or female rats during the 28-day experimental period.This suggests that the tested doses (50, 75, 100, and 150 mg/kg) presented low toxicity.In previous studies with mice, higher doses (300, 350, and 400 mg/kg) of the mixture, showed clear signs of toxicity including death, especially in males (Schmitt et al., 2012).
Body weight did not significantly change during the 28-day toxicity test.However, it was possible to observe that body weight gain was higher in females than males (Figure 1).Necropsy showed no changes in relative organ weights and no macroscopic alterations in either male or female rats.
Animals did not lose weight in contrast to the purported aims of the products.Interestingly, the results revealed a different profile between genders.Females showed more body weight gain, demonstrating them as less susceptible to the effects of the mixture.This fact could be related to hormonal differences between males and females; further studies focusing on hormones could elucidate this point.
Hematological and biochemical analysis results are presented in Table I.No significantly alterations were observed in male or female rat hematological parameters.Biochemical analysis revealed increases (p < 0.05) in AST, ALT, and creatinine levels in male rats treated with 100 and 150 mg/kg of the mixture.However, there was a significant decrease (p < 0.05) in AST for female groups treated with 100 and 150 mg/kg of the mixture.
Males displayed an upward trend in both AST and ALT enzyme levels as dose increased.According to adopted reference values, AST is normal whereas ALT is increased and may indicate some liver damage.In the same way, creatinine levels in all male treated groups showed a progressive increase, which was significantly higher in the 100 and 150 mg/kg treated groups compared to controls (p < 0.05).It was also outside the normal range (0.2-0.6 mg/dL) for male Wistar rats (Giknis, Clifford, 2008) indicating possible renal damage.Although there is undeniable evidence of the nephrotoxic effects of the mixture, the mechanism responsible is unclear.It may be caused by a similar mechanism to amphetamines, whose vasoconstrictor properties predispose the renal medulla to ischemia and hypoxia.In addition, there could be direct toxic effects from the drugs and/or their metabolites, as well as enhanced oxidative stress in the kidney (Carvalho et al., 2012).
The liver is a target for amphetamines and their analogues (as in the case of synephrine and ephedrine) since they are biotransformed primarily in this organ, as is caffeine.During the biotransformation process (e.g.3,4-Methylenedioxy-methamphetamine -MDMA,  a hallucinogenic amphetamine) free radicals can be generated (Carvalho et al., 2004) and cause liver damage.Custodio et al. (2010) studied mitochondria involvement in MDMA and 4-methylthioamphetamine (MTA) produced liver toxicity and found that, depending on dose, both amphetamine derivatives (especially MTA in high concentrations) cause mitochondrial dysfunction, which is another mechanism for hepatotoxicity.Hepatic enzyme alterations in female groups were opposite to those in male groups; their AST values significantly decreased in the 100 and 150 mg/kg treated groups related to controls (p < 0.01).However, the values were within normal limits for female Wistar rats (63-175 U/L) (Giknis, Clifford, 2008).
There are many reports of gender being a critical factor in drug-induced liver injury (Hemieda, 2007;McConnachie et al., 2007;Dever, Elfarra, 2008).Moreover, most of those reports demonstrate that males were more susceptible to hepato toxin-induced liver injury than females.Studies have reported that bioactivation and estrogen concentration are mainly responsible for the gender-dependent discrepancy in liver injury (McConnachie et al., 2007;Dever, Elfarra, 2008;Liang et al., 2011).Also, gender differences in pharmacokinetics and pharmacodynamics characterize many drugs and contribute to individual differences in drug efficacy and toxicity.Drug metabolism is the primary cause of genderdependent pharmacokinetics and reflects underlying gender differences in the expression of hepatic enzymes active in the metabolism of drugs, steroids, fatty acids, and environmental chemicals, including cytochrome P450 enzymes (P450s), sulfotransferases, glutathione transferases, and UDP-glucuronosyl transferases.Studies in rat and mouse liver models have identified more than 1000 genes whose expression is gender-dependent; together, these genes impart substantial sexual dimorphism on liver metabolic function and pathophysiology (Waxman, Holloway, 2009).Arbo et al., (2009a) tested Citrus aurantium extracts (400, 2000, or 4000 mg/kg corresponding to 30, 150, and 300 mg/kg of p-synephrine, respectively) and p-synephrine (30 and 300 mg/kg) in male mice daily for 28 days and found no changes in biochemical parameters ALT, AST, or creatinine.This result can in fact corroborate that associating p-synephrine with others substances promotes physiological alterations, as the doses of the tested mixture were 50, 75, 100, and 150 mg/kg, corresponding to only 5, 7.5, 10, and 15 mg/kg of p-synephrine.
Oxidative stress evaluations are presented in Table II.Male rats treated with 100 and 150 mg/kg showed significant increases (p < 0.05) of 51% and 62% respectively in MDA values and a decrease in GSH levels in all treated groups.GPx activity in females showed a significant increase of 58% in the 75 mg/kg treated group compared to controls.
Oxidative stress biomarker evaluation indicated the occurrence of lipid peroxidation which is characterized by increased MDA (Grotto et al., 2009).It is known that amphetamines and analogues increase the production of reactive species such as free radicals (Giovanni et al., 1995;Colado et al., 1997;Fleckenstein et al., 1997;Yamamoto, Zhu, 1998).Evidence shows that reactive oxygen species (ROS) directly or indirectly contribute to amphetamine toxicity mechanisms (Kovacic, Cooksy, 2005), and although the toxic effects caused by abusive consumption of these substances is already known, the molecular factors that contribute to these effects are not completely understood (Frey et al., 2006).
Both p-synephrine and ephedrine are structurally and pharmacologically related to amphetamine (Firenzuoli et al., 2005), which could be associated to the oxidative stress seen in this study.
Caffeine-derived effects could favor the production of free radicals and a subsequent increase in oxidative stress such as the metabolic inactivation of catecholamines (Halliwell, Gutteridge, 1985;Jewett et al., 1989) and the increase in oxidative metabolism (Shigenaga et al., 1994;Thompson et al., 2001) including its own hepatic metabolism (Vistisen et al., 1992).There are also reports suggesting that caffeine is capable of inducing certain forms of oxidative damage by increasing lipid peroxidation (Dianzani et al., 1991).On the other hand, caffeine has been reported as a protective substance in cellular damage (Kamat et al., 2000;Krisko et al., 2005) with beneficial antioxidant effects (Nikolic et al., 2003) probably due to the main metabolites of caffeine, 1-methylxanthine and 1-methyluric acid, which are highly effective antioxidants (Lee, 2000).So, the real behavior of caffeine is unclear.
There are no studies focusing on oxidative stress related to salicin, but aspirin (acetylsalicylic acid, which belongs to the same salicylate family as salicin) showed direct endothelial protection from oxidative damage at antithrombotic concentrations and demonstrated antioxidant and radical scavenging properties (Podhaisky et al., 1997).
In addition, the oxidative stress seen in the 100 and 150 mg/kg male treated groups is confirmed by a decrease in GSH levels in all male treated groups.One of the most important roles of the GSH antioxidant system is the detoxification of endogenously generated peroxides.GSH production is considered the first line of defense against oxidative damage and free radical generation, where GSH functions as a scavenger and co-factor in metabolic detoxification (Nozal et al., 1997).GSH levels increased in mice treated orally for 28 consecutive days with p-synephrine (30 and 300 mg/kg) and with C. aurantium extracts (4000 mg/kg corresponding to 300 mg/kg of p-synephrine respectively) (Arbo et al., 2009a).
Interestingly, oxidative stress was not apparent in female rats.There were no changes in MDA or GSH levels with the treatment received.Some studies have reported gender differences in antioxidant capacity (Yamamoto et al., 2002;Ilhan et al., 2004) which could account for this finding.There is substantial evidence suggesting that the female hormone estrogen has potent antioxidant properties while the male steroid testosterone possesses no antioxidant activity (Yagi, Komura, 1986).Sugioka et al., (1987) reported that in contrast to other natural steroids, estrogens have a hydroxyl group in their structure which is also found in Vitamin E. Thus, estrogen may have the potential to quench peroxy radicals and terminate peroxidative chain reactions (Tiidus, 2000).According to Ayres et al. (1998), at near physiological concentrations in vitro, estrogen may indeed be able to directly inhibit superoxide production and act as a peroxidation chain breaking antioxidant for both lipids and DNA.Huang and colleagues (1999) noted the potential for estrogen to reduce oxidation of low density lipoproteins.In addition, some studies have shown that gender differences between male and female also exist in the antioxidant status of normal animals, including differences in Vitamin C and E levels in rats (Tiidus et al., 1999).
Gender may also affect liver glutathione status, and male rats have approximately 50% more reduced and total glutathione than females (Tiidus et al., 1999), while muscle activities of antioxidant enzymes superoxide dismutase, catalase, and glutathione peroxidase appear to be similar between genders in rats (Tiidus, 2000).Nevertheless, the results of our study showed a significant decrease in male rat GSH status, whereas no alterations were seen in their female counterparts.This fact could be explained as an organism response to oxidative damage, shown by MDA levels, as the function of GSH is to defend against oxidative damage and free radical generation (Nozal et al., 1997).
Alterations in GPx activity in the female 75 mg/kg group should be not considered as it did not show a linear profile.According to Lewis et al. (2002), some factors can be useful in differentiating between a significant change from control values and a treatment-related effect.This difference is less likely to be an effect of treatment if: there is no obvious dose response; it is due to findings in one or more animals that could be considered outliers; or it is normal biological variation (within reference value limits).

CONCLUSION
In this experimental work we evaluated a 28-day period of oral exposure to a mixture of p-synephrine, ephedrine, salicin, and caffeine, which has been used in weight loss formulations.Our findings indicate that the continued use of the mixture can produce changes in hepatic and kidney function and in oxidative stress biomarkers; these are more pronounced in males than females.More studies are necessary to clarify gender differences in sub-chronic toxicity profile and the protector role of estrogen against the effect of the mixture in females.
Further investigations should include long-term toxicity studies to better explain the effects of the mixture, as well as determine other oxidative stress and biochemical (hepatic and renal) markers.Products containing this mixture should be used with caution until the toxicological profile has been fully elucidated, whereas the number of toxicological accidents related to the use of dietary supplements whose formulations often vary, is very frequent.

FIGURE 1 -
FIGURE 1 -Relative body weight gain in Wistar rats male (A) and female (B) after oral treatment for 28 consecutive days with p-synephrine associated to ephedrine, salicin and caffeine (10:4:6:80 w/w).Each point represent the mean ± standard error of the mean (n=8).

TABLE I -
Hematological and plasmatic biochemical parameters evaluated in male and female Wistar rats after 28 days of treatment with a mixture of p-synephrine, ephedrine, salicin and caffeine (10:4:6:80 w/w) Data are expressed as mean ± standard error of the mean (n=6-8 males and 6-8 females per group).*Significantly different from their respective gender control group (p < 0.01) by ANOVA/Bonferroni.AST: aspartate aminotransferase; ALT: alanine aminotransferase

TABLE II -
Oxidative stress biomarkers evaluated in male and female Wistar rats after 28 days of oral treatment with a mixture of p-synephrine, ephedrine, salicin and caffeine (10:4:6:80 w/w)