Passiflora incarnata treatment during gestation and lactation : toxicological and antioxidant evaluation in wistar dams

1Pharmacy Division, University Hospital, State University of Londrina, UEL, Londrina, PR, Brazil, 2Research Laboratory, Health Sciences Center, State University of Londrina, UEL, Londrina, PR, Brazil, 3Department of Pathology, Clinical and Toxicological Analyses, Health Sciences Center, State University of Londrina, UEL, Londrina, PR, Brazil, 4Department of Physiological Sciences, Biological Sciences Center, State University of Londrina, UEL, Londrina, PR, Brazil

Passiflora incarnata is marketed in many countries as a phytomedicine.Even though the directions of most marketed phytomedicines recommend them to be used under medical supervision, reproductive and developmental studies are sparse and not mandatory for regulatory purposes.In this study, a reproductive toxicity evaluation of P. incarnata was conducted in Wistar rats gavaged (30 or 300 mg/kg) during pregnancy and lactation.Moreover, considering that antioxidant properties have been attributed to flavonoids present in the genus Passiflora, it was also evaluated the antioxidant/pro-oxidant balance in the plasma of these dams and the antioxidant potential in an in vitro test.P. incarnata treatment did not influence dams´ body weight as well as reproductive (gestation length, post-implantation loss, litter size, litter weight) and hepatic (albumin, AST, ALT, GGT) parameters.The antioxidant property of P. incarnata was evidenced both in vivo (increase in the total antioxidant plasmatic potential) and in vitro (decrease in neutrophil-induced respiratory burst).The results from the present study indicate that under the experimental conditions evaluated, P. incarnata treatment during gestation and lactation presented antioxidant activity in the absence of maternal reproductive toxicity.
Passiflora incarnata is marketed in many countries as an anxiolytic and sedative phytomedicine (Soulimani et al., 1997;Zanoli, Avallone, Baraldi, 2000) and its mechanism of action involves potentiation of the GABAergic system (Zanoli, Avallone, Baraldi, 2000;Grundmann et al., 2008).As observed for many HMP used by the population, information regarding the safety of P. incarnata use during pregnancy and/or lactation is sparse (Veríssimo et al., 2011).To the best of our knowledge, only one study suggested, in vitro, an uterotonic action of this plant (Ruggy, Smith, 1940).Based on this study, the labels of P. incarnata-based phytomedicines marketed in Brazil indicate that its use during pregnancy and/or breastfeeding needs physician or dentist supervision (Brasil, 2008).However, advising without scientific support certainly is not an easy task for these professionals.
This study was designed to investigate if P. incarnata induces maternal toxicity when administered to Wistar rats during pregnancy and lactation.Moreover, considering that antioxidant properties have been attributed to flavonoids present in the genus Passiflora (Rudnicki et al., 2007;Masteikova et al., 2008), it was also evaluated the antioxidant/pro-oxidant balance in the plasma of these dams as well as P. incarnata antioxidant potential through an in vitro test.

Animals and treatment
Male and female wistar rats (75-90 days) from the colony of the State University of Londrina (UEL) were mated (2 or 3 females and 1 male per cage) overnight and gestational day (GD) 0 was considered if there were sperm and estrus phase cells in vaginal smears.
Pregnant rats were housed singly and randomly divided into control-or P. incarnata-treated dams.Control dams (Con: 15 dams) were gavaged daily with tap water whereas P. incarnata were gavaged with 30 (P30: 17 dams) or 300 (P300: 14 dams) mg/kg.Treatment lasted from GD 0 to lactational day (LD) 21 and doses were daily adjusted according to weight.A HMP marketed in Brazil (Passiene™, Herbarium Laboratories) was employed as the source of P. incarnata.This syrup is standardized in 0.19% of vitexin.In humans, the dose of 20-30 mg/kg/day is usually recommended.
All animals had free access to water and regular lab chow (Nuvital™).They were maintained in the vivarium of the Department of Physiological Sciences at 21±2 °C on a 12:12h light-dark cycle (on at 0700).All animal procedures were approved by the UEL Ethics Committee for Animal Research (CEEA 16/2010).

Maternal toxicity and reproductive performance of dams
Maternal body weight was measured daily during gestation and lactation.Furthermore, the corrected maternal body weight gain was estimated subtracting the weight on GD 0 from the weight on LD 0. On LD 0, the number of pups in each litter was determined as well as the litter weight.On LD 21, dams were submitted to an open-field in order to evaluate general activity (Bacchi et al., 2013).They were euthanized with diethyl ether and their livers were dissected and weighed.The uterine horns were also dissected and the numbers of implantation sites were counted to determine the rate of postimplantation loss [(number of implantations-number of live fetuses/ number of implantations)x100].Blood was collected through cardiac puncture and centrifuged at 1000 g for 15 minutes.Plasma was aliquoted and stored at -70 ºC for subsequent laboratory testing.Hepatic function was evaluated through the determination of gamma-glutamyl transferase (GGT), aspartate aminotransferase (AST), alanine aminotransferase (ALT), albumin and total proteins.GGT, ALT and AST were analyzed using UVkinetic methods in an automated biochemical system (Dimension ® RXL, Deerfield, IL, USA).Albumin and total protein were measured through the use of colorimetric commercial kits (Ebram™, Brazil).

Plasmatic antioxidant/pro-oxidant balance
Total serum antioxidant potential (TRAP) was determined by chemiluminescence using an adaptation from the method described by Repetto et al. (1996).This experiment was conducted in a Beckman b counter, model LS 6000 (Fullerton, CA, USA) in a non-coincident counting mode for 25 minutes and with a response range between 300 and 620 nm.The system was calibrated with vitamin E analogue (Trolox).A comparison of the induction time after the addition of known concentrations of Trolox and plasma allows obtaining TRAP values of Trolox equivalents.
Pro-oxidant markers evaluated in this work were lipid hydroperoxides, malondialdehyde (MDA), products of advanced protein oxidation (AOPP) and nitric oxide (NO).
L i p i d h y d r o p e r o x i d e s w e r e m e a s u r e d b y spectrophotometry (FOX) using an adaptation of the technique described by Jiang, Woollard, Wolff (1991).The method is based on the oxidation of ferrous to ferric ions by peroxide under acidic conditions, which reacts with the indicator dye (xylenol orange) and produces a colored complex.The system was calibrated with a standard solution of 1,1,3,3-tetraethoxyproprane.The reading was performed in a spectrophotometer (Helios α ThermoSpectronic ™, Waltham, MA, USA) at a wavelength of 560 nm and the results are expressed in mM.
MDA, which results from the decomposition of hydroperoxides, was quantified by high performance liquid chromatography using an adaptation of the technique described by Bastos et al. (2012).Results are expressed in nM/mg of protein.
AOPP was quantified by the method described by Witko-Sarsat et al. (1998).The method is based on the reaction of oxidized proteins with potassium iodide in acid conditions.The calibration curve was obtained from a stock solution of 1mM chloramine and the absorbance of the reaction was determined spectrophotometrically (Helios α ThermoSpectronic ® , Waltham, MA, USA) at 340 nm.AOPP concentrations are expressed in μM/L equivalent of chloramine.
NO levels were assessed indirectly by determining the plasma concentration of nitrite, using an adaptation of the technique described by Navarro-Gonzalez, Garcia-Benayas and Arenas (1998).The quantification of nitric oxide was made in a microplate reader (Biochrom ASYS Expert Plus™, Holliston, MA, USA) at a wavelength of 550nm.The results are expressed in µM/L.

In vitro evaluation of the antioxidant activity of P. incarnata
The reactive oxygen species (ROS) production by neutrophils (respiratory burst) was evaluted by chemiluminescence according to an adaptation of the method described by Freitas et al. (2008) and Huber, Krötz-Fahning, Hock (2006) in a multiplate reader (Victor X3, Perkin Elmer, USA).Human neutrophils were isolated from whole blood through gradient density centrifugation.Neutrophil burst was induced by phorbol myristate acetate (PMA) in the presence of 5 or 10 µL of P. incarnata syrup (75 mg/mL) or vehicle (base syrup).The detection probe was luminol.Kinetic readings were initiated immediately after cell stimulation and measured each other minute for 60 minutes.Effects are expressed as chemiluminescence arbitrary units (cpm).For each experimental group, 8-10 replicates were conducted.For statistical analysis, the peak value of each curve was used, independently of the time it occurred.

Statistical analysis
Initially, an exploratory analysis was conducted to evaluate normal distribution (Shapiro-Wilk test) and homogeneity of variance (Levene test) of each variable.For variables that presented normal distribution and homogeneity of variance, parametric analysis were conducted.Due to the high number of dams employed in this study, they could not be treated all at once and our sample is composed from animals belonging to two different cohorts.Because of this, cohort was added as a factor in the statistical analysis.The parametric statistical tests employed in this study were:

Maternal toxicity and reproductive performance of dams
Gestation length was not influenced by Passiflora treatment and no clinical signs of toxicity were observed during the daily treatment.RMANOVA indicated an effect of the day on dams' weight [F(3,111)=651.9, p<0.0001] reflecting the fluctuations that occur during pregnancy and lactation (data not shown).P. incarnata treatment did not influence (p>0.05)weight gain as well as the corrected weight gain of dams during gestation (weight in LD 0weight in GD 0).Means and SEM for the corrected weight gain were Con=32.7±4.2 (n=15); P30=25.9±3.2 (n=17); P300=22.6±2.8 (n=14).Two-way ANOVA showed that P. incarnata treatment did not influence (p>0.05) the number of uterine implantation sites, post-implantation loss, number of pups per litter and weight of pups at birth (Table I).Dead pups at birth were observed in 2 Con litters and in 2 P300 litters.No macroscopic alteration was observed during necropsy.

In vivo evaluation of the antioxidant activity of P. incarnata
Data from the antioxidant/pro-oxidant plasmatic balance in dams are presented in Table III.Two-way ANOVA indicated that P. incarnata treatment influenced TRAP levels [F(2,42)=5.1, p<0.05].Bonferroni post-hoc test revealed that, compared to CON, P30 tended (p=0.06) to increase TRAP and P300 significantly increased it (p<0.05).Even though total plasmatic antioxidant capacity was increased in P. incarnata-treated dams, a significant difference in the levels of the pro-oxidant markers evaluated (lipid hydroperoxides, MDA, AOPP and NO) was not detected (Two-way ANOVA, p>0.05).

In vitro evaluation of the antioxidant activity of P. incarnata
Figure 1A presents the chemiluminescence produced by PMA-stimulated neutrophils in the absence (Control) or presence of 5 and 10 µL of P. incarnata syrup (75 mg/ mL) or vehicle (10 µL of base syrup).Each curve is the mean of 8-10 replicates.

DISCUSSION
In reproductive and developmental toxicity studies, evaluation of maternal toxicity is critical.This study was conducted according to the International Conference on Harmonization, Technical Requirements for Registration of Pharmaceuticals for Human Use guideline (ICH, 2005).The P. incarnata regimen of treatment adopted in the present study did not result in maternal toxicity as evidenced by the lack of effect on parameters of overt general toxicity (clinical signs, weight gain, general activity), hepatic function and reproductive performance.Literature lacks toxicological studies conducted in animals treated repeatedly with P. incarnata and none of the available monographs of P. incarnata mention results from these types of studies.
In this work, the antioxidant property of P. incarnata was evidenced both in vivo and in vitro.In vivo, there was a dose-dependent increase in TRAP which reflects both hydro and lipossoluble antioxidant substances (Repetto et al., 1996).In vitro, there was a decrease in PMA-induced respiratory burst in neutrophils, suggesting a scavenger activity of the syrup.In vitro antioxidant activity has also been described for aqueous and ethanolic extracts of P. incarnata in the DPPH (2.2-Diphenyl-1-picrylhydrazide) and ABTS (Azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid) methods (Masteikova et al., 2008).Flavonoids have been described as the main phytoconstituents of P. incarnata (Raffaelli, Moneti, Toja, 1997) and the antioxidant activity of this species has been attributed to these molecules (Masteikova et al., 2008).Flavonoids are phenolic substances that can stabilize and delocalize the unpaired electron within its aromatic ring and are able to reduce free radical formation and to scavenge free radicals.Among the flavonoids that have been described in P. incarnata, vicenin, isovitexin and orentin have been shown to play a major role in the in vitro antiradical activity of this species (Masteikova et al., 2008).Moreover, in an in vivo study conducted with Wistar rats gavaged with vitexin and isovitexin, increased total antioxidant capacity has been described (Cao et al., 2011).
Although total plasmatic antioxidant potential was found to be increased in dams treated with P. incarnata, it was not observed a significant decrease in the plasmatic markers of oxidation, i.e., lipid hydroperoxides, MDA, AOPP and NO.We cannot offer a definitive explanation for this observation but it could be hypothesized that an increased antioxidant capacity could be important to protect macromolecules from oxidation in the presence of oxidative stressors and not at baseline condition.Similar observation, i.e., increased TRAP in normal subjects in the absence of altered levels of lipid hydroperoxides, has also been described in a study from our group designed to evaluate the impact of green tea consumption in normal and dyslipidemic humans (Camargo, Daguer, Barbosa, 2006).
It is interesting to point out that, to our knowledge, this is the first study to describe in vivo antioxidant activity for P. incarnata.In the literature, decreased markers of oxidation have been described with other species from the Passiflora genus.P. edulis juice decreased levels of TBARS in the blood (reduced lipid peroxidation) of male rats treated for 28 days with 1,000mg/kg of the juice (De Souza et al., 2012).P. alata leaf extract (1 and 5 mg/kg, gavage, 30 days) prevented the increase of hepatic TBARS after a challenge with carbon tetrachloride in male rats (Rudnick et al., 2007).
In conclusion, the results from the present study indicate that P. incarnata treatment during gestation and lactation presents antioxidant activity in the absence of maternal reproductive toxicity.

FIGURE 1 -
FIGURE 1 -Activation of human neutrophils by PMA in the absence (control) or presence of P. incarnata (5 or10 µL) or base syrup (10 µL).Each group was composed by 8-10 replicates. A. Kinetic curves obtained each other minute for 60 minutes.B. Box plot of the peak values from the kinetic curves.Data were analyzed by Kruskal-Wallis complemented with Dunn´s test.* p<0.05 compared to CON; # p<0.05 compared to base syrup.

TABLE I -
Reproductive parameters and general activity in the open-field of dams treated with P. incarnata during gestation and lactation

TABLE II -
Plasmatic parameters of hepatic function in dams treated with P. incarnata during gestation and lactation

TABLE III -
Plasmatic oxidative stress parameters in dams treated with P. incarnata during gestation and lactation