1 Introduction

An epileptic seizure is a kind of brain dysfunction caused by over discharge. In the injured brain, endothelial cells release cytokines, activating T cells and triggering acquired immune disorders, thus leading to the pathogenesis of epilepsy (Huang and Zhu, 2018). Damage of blood–brain barriers (BBB) causes entry of inflammatory immune cells. Activation and neutrophil recruitment by kinin lead to mitochondrial alterations, microglial activation and generation of reactive oxygen species (ROS). ROS, in turn, cause oxidative damage, which alters the cellular tight junctions. In addition, other animal studies revealed that breakdown of BBB leads to transcriptional changes in the neurovascular connections and eventual neurodegeneration (Shlosberg et al., 2010). The inflammatory processes represent key contributors to acute and chronic neurological disorders, such as epilepsy (Poole et al., 2000). IL-1β plays a role in the initiation and progression of neuronal dysfunction during epilepsy.

The antiepileptic drugs (AEDs) of epilepsy is associated with dose-related and chronic toxicity, and teratogenic effects (Poole et al., 2000; Ropper, 2005). Drugs based on natural products and folk remedies can offer safe and efficient alternative for AEDs (Raza et al., 2003).

Passiflora species have gained worldwide prominence because of their varied content of phyto-constituents of high therapeutic value that represent potential raw material for the development of new drugs. Amongst the 500 species of the genus Passiflora, Passiflora incarnata (P. incarnate) is the one that has extensive clinical applications throughout the world. P. incarnata Linneaus possesses significant CNS depressant properties (Dhawan et al., 2004). The drug is highly effective in convulsions when given prior to an approaching attack. P. incarnata is praised for its control over meningeal inflammations and their consequent spasms of childhood (Dhawan et al., 2004).

Various phytoconstituents were reported to be present in P. incarnata including carbohydrates)Dhawan et al., 2004), amino acids, and a cyanogenic glycoside gyanocardin. In addition, monoflavonoid of Passiflora sp. was reported to reduce morphine withdrawal in vitro (Capasso et al., 1998). Chrysin (5,7-dihydroxyflavone), one of the monoflavonoid found in Passiflora sp., has been used as an anxiolytic anticonvulsant)Zanoli et al., 2000). Chrysin, a natural product found in a variety of flowers and fruits, has received attention because of its potent antioxidant and anti-inflammatory properties (Ahad et al., 2014; Hajimohammadi and Nosrati, 2018).

Despite the well-documented phytochemical reports on P. incarnata, the exact mode of its anti-inflammatory and antioxidant effects and the phyto-constituents responsible for its much-acclaimed effects have never been described clearly. Moreover, only few studies focusing on the potential role of P. incarnata as an anti-inflammatory and antioxidant herbal food supplement in epileptic models are available. The study aims at assessing the ameliorative changes after treatment with P. incarnata in case of pilocarpine-induced epilepsy in male albino rats.

2 Materials and methods

3 Results

3.2

3.2 Changes in brain electrolytes

Regarding the changes in brain electrolytes, pilocarpine injection caused dramatic decrease in K⁺, Mg⁺⁺ and Ca⁺⁺ levels in brain tissues, recording 3.05 ± 0.08 mmol/L; 0.29 ± 0.08 mg/dL and 8.00 ± 0.25 mg/dL, respectively (Fig. 1). In addition, Na⁺ recorded the highest level in the epileptic non-treated group (189.32 ± 7.62 mmol/L). These imbalanced levels were corrected after treatment with Passiflora incarnata extract (200 mg/kg); and recorded 164.66 ± 5.27 mmol/L; 4.07 ± 0.47 mmol/L; 0.698 ± 0.04 mg/dL and 8.63 ± 0.21 mg/dL for Na⁺; K⁺, Mg⁺⁺ and Ca⁺⁺, respectively.

Close

Fig. 1

Changes in sera electrolytes of different groups: Control (C); Epileptic-non treated group (E); Passiflora incarnata group (PI) and Epileptic-treated with P. incarnata; (E-PI) (200 mg/Kg. b.wt). Groups not sharing common superscripts denote significant differences (*P < 0.05, **P < 0.01 and ***P < 0.001). Values were represented as Mean ± SD; n = 8 rats.

Export to PPT

In addition, Fig. 2 shows a significant decrease (P < 0.001) in the level of Na⁺/K⁺-ATPase in brain tissues (an electrogenic transmembrane ATPase) after induction of status epilepsy, which recorded 2.11 ± 0.17 mmol/ml. The treatment with P. incarnata remodified Na⁺/K⁺-ATPase to the normal level 4.57 ± 0.34 mmol/ml as recorded in the control group (4.44 ± 0.38 mmol/ml). Moreover, creatine kinase (CK) showed a highly significant increase of 294.35 ± 12.53U/L in the second group (E) and returned to 183.72 ± 5.34 U/L after treatment with P. incarnata, though still significantly higher than the control group (111.23 ± 5.92 U/L).

Close

Fig. 2

Changes in brain energy and neurotransmitters of different groups: Control (C); Epileptic-non treated group (E); Passiflora incarnata group (PI) and Epileptic-treated with P. incarnata; (E-PI) (200 mg/Kg. b.wt). Groups not sharing common superscripts denote significant differences (*P < 0.05, **P < 0.01 and ***P < 0.001). Values were represented as Mean ± SD; n = 8 rats.

Export to PPT

3.3

3.3 Changes in neurotransmitters

In addition, the present data revealed significant decrease (P < 0.001) in different neurotransmitters and classical monoamines, e.g. norepinephrine (NE) and epinephrine (EP), whereas they decreased in the epileptic non-treated group, recording levels of 43.17 ± 5.75 and 32.10 ± 5.37 Pg/ml, respectively. Treatment with P. incarnata mitigated and upregulated them again to the healthy levels of 67.37 ± 4.62 and 48.65 ± 3.59 for norepinephrine and epinephrine, respectively (Fig. 2).

Regarding changes in other monoamines, e.g. dopamine (DOPA) and serotonin (5-HT), the epileptic non-treated group demonstrated a significant increase (P < 0.001) in DOPA (57.70 ± 3.77 µg/ml) and a significant decrease (P < 0.001) in serotonin level (39.78 ± 2.56 ng/ml), as compared to the control group, which recorded 24.60 ± 2.36 µg/ml and 77.07 ± 7.90 ng/ml for DOPA and serotonin, respectively (Fig. 3). Moreover, the treatment with P. incarnata modified both DOPA and serotonin levels, approximating their normal values of 37.43 ± 3.53 µg/ml and 67.72 ± 4.05 ng/ml, respectively (Fig. 3).

Close

Fig. 3

Changes in neurotransmitters (monoamines) and some amino acids of different groups: Control (C); Epileptic-non treated group (E); Passiflora incarnata group (PI) and Epileptic-treated with P. incarnata; (E-PI) (200 mg/Kg. b.wt). Groups not sharing common superscripts denote significant differences (*P < 0.05, **P < 0.01 and ***P < 0.001). Values were represented as Mean ± SD; n = 8 rats.

Export to PPT

The effects of status epilepticus on the brain concentrations of glutamate, gamma-aminobutyric acid (GABA), aspartate and glycine were measured, as illustrated in Fig. 3. As mentioned in the epileptic non-treated group, GABA and glycine showed significant decrease level (13.82 ± 2.17 pg/ml and 4.13 ± 0.88 μg/ml, respectively), whereas glutamate and aspartic acid levels showed significant increase (14.58 ± 1.02 μg/ml and 13.60 ± 0.90 ng/ml, respectively). Treatment with P. incarnata modified the imbalanced levels of glutamate, γ-aminobutyric acid (GABA), aspartate and glycine to normal values of 8.165 ± 0.613 μg/ml; 25.338 ± 1.601 pg/ml; 9.022 ± 0.621 ng/ml and 8.000 ± 0.947 μg/ml, respectively.

4 Phenotyping of CD4⁺ and CD8⁺ brain infiltrative cells

Meanwhile, phenotyping of CD4⁺ and CD8⁺ brain infiltrative cells is illustrated in Table 2 and Fig. 4 by FACS-analysis. Changes in CD4⁺ and CD8⁺ markers in brain-infiltrating cells showed a highly significant (P < 0.001) elevation in both CD4⁺ and CD8⁺ expressed cells in the second group (E) (P < 0.001), recording values of 54.13 ± 4.54% and 36.52 ± 3.08%, respectively. However, treatment with P. incarnata caused a diminution in the subpopulation of CD4 + and CD8 lymphocytes and corrected the above-mentioned activities, recording 27.89 ± 2.31% and 17.31 ± 1.20% for CD4⁺ and CD8⁺, respectively. The modified activity was compared to the control group (C) which recorded 17.02 ± 2.63% and 11.55 ± 1.29% for CD4⁺ and CD8⁺, respectively.

Close

Fig. 4

Representative dot plots of FACS-analysis showing changes in CD4 + and CD8 + brain infiltrating lymphocytes in different animal groups.

Export to PPT

5 Assessment of the proinflammatory cytokines

Concerning assessment of the proinflammatory cytokines’ level, the ameroliative levels of IL-1β, IL-6, IL-17A, TNF-α, and TGF-β were significantly (P < 0.001) reduced to 30.03 ± 5.02; 59.72 ± 7.42; 69.42 ± 2.66; 53.37 ± 1.74 and 74.76 ± 5.30 pg/mL, respectively, after treatment with P. incarnata (Fig. 5). These ameroliative changes were compared to their level in the epileptic non-treated group (92.40 ± 7.34; 114.68 ± 2.40; 94.03 ± 4.27; 108.00 ± 6.27 and 124.70 ± 3.10 pg/mL, respectively). In addition, the treatment with Passiflora extract (200 mg/kg) showed an ameroliative increase in anti-inflammatory cytokines, e.g. IL-10 and IL-13, which re-increased significantly (P < 0.001) in the epileptic-treated group with P. incarnata, recording 67.17 ± 4.43 and 93.88 ± 9.27 pg/mL, respectively.

Close

Fig. 5

Changes in plasma pro and anti-inflammatory cytokines of different groups: Control (C); Epileptic-non treated group (E); Passiflora incarnata group (PI) and Epileptic-treated with P. incarnata; (E-PI) (200 mg/Kg. b.wt). Groups not sharing common superscripts denote significant differences (*P < 0.05, **P < 0.01 and ***P < 0.001). Values were represented as Mean ± SD; n = 8 rats.

Export to PPT

6 Discussion

The present results showed that the antioxidant effect of P. incarnata treatment came in agreement with many reports (de Carvalho et al., 2011; Elsas et al., 2010; Sena et al., 2009). It is noteworthy that the antioxidant activities of Passiflora spp. can be traced to its nutritional components of flavonoids and phenols, as well as cysteine, glutathione, ascorbic acid, tocopherol, tannins, and aromatic amines (Saravanan et al., 2014). Generally, flavonoids can be oxidized by radicals, resulting in more stable, less-reactive radicals. Some flavonoids can scavenge superoxides, while others can scavenge the highly reactive oxygen-derived radical peroxynitrite (Esposito et al., 2002; Nabavi et al., 2015). In addition, Ingale and Kasture (2017) referred to the antioxidant potential of Passiflora via significant DPPH (2,2-diphenyl-1-picrylhydrazyl) and H₂O₂ scavenging ability due to the content of phenolic compounds, mainly flavonoids.

Moreover, Sasikala et al. (2014) observed a highly antioxidant scavenging capacity of Passiflora extracts spp. towards free radicals. These properties can reduce and donate hydrogen ions and mitigate side effects of free radicals released during lipid peroxidation and cell damage. In addition, Saravanan et al. (2014) confirmed that extracts of Passiflora spp. could donate electrons to the released free radicals in biological tissues and reduce its inflammatory stress. Also, Nabavi et al. (2015) mentioned that chrysin, as one of the flavonoids component of P. incranata, can reduce nerve cell damage via preventing the release of nitric oxide and inflammatory cytokines from activated microglia, hence preventing neurodegeneration events.

Furthermore, Colomeu et al. (2017) reported that phenolic compounds are ubiquitously present in plants with antioxidant properties. These activities may be attributed to their redox properties, e.g. hydrogen donors, reducing agents, singlet oxygen quenchers and metal chelators.

The significant increment (P < 0.001) in the levels of electrolytes recorded throughout the present data could be explained by Liu et al. (2008) who reported significant content of electrolytes and minerals in pasipay (P. incarnata), e.g. Na⁺, K⁺, Mg⁺² and Ca⁺². Therefore, they suggested such herbal drug as supplement food source in pharmaceutical and industrial fields.

In accordance with our data, during epileptic episodes, a massive increase of amino acids and neurotransmitters is observed (Bramlett and Dietrich 2004). These molecules lead to increased Ca²⁺, Na⁺ and K⁺ fluxes via stimulation of glutamate receptors. As a result, catabolic processes will be activated, resulting in breakdown of the blood–brain barriers (BBB) (Werner and Engelhard 2007). Furthermore, chrysin, as the main plant extract and bioactive component content of P. incarnata, can modify Na⁺, K⁺-ATPase activity in the prefrontal cortex and hippocampus of mice (Borges et al., 2016).

Previously, Singh et al. (2012) demonstrated that rats treated with extracts of P. incarnata showed a significant treatment of seizure severity and immobility period in a dose- and time-dependent manner. These effects may be traced to the retained levels of serotonin and noradrenaline in the brain.

The recorded changes in brain amino acid and neurotransmitters levels are strikingly similar to those observed in other studies. The current anticonvulsant effects of pasipay (P. incarnata) may be related to its activation for benzodiazepine receptor (BZD), GABAergic and opioid systems (Fernández et al., 2006). Nassiri-Asl et al. (2007) revealed that a 100% protection for seizure episodes and mortality occurs after treatment with a dose of 0.4 mg/kg of P. incarnata.

Because GABA is a major inhibitory neurotransmitter, the present data revealed that P. incarnata increased the level of GABA. This is in accordance with the findings of Appel et al. (2011) and Guerrero and Medina (2017) who noticed that P. incarnata bioactivities may be mediated by GABA modulation by activation of the kappa opioid receptor (KOPr) that leads to support of GABAergic activity or attenuation of glutamatergic activity. This may confirm that P. incarnata could activate KOPr and result in protective effects against pentylentetrazole (PTZ)-induced seizure (Nassiri-Asl et al., 2007). The presence of GABA has been reported in P. incarnata and has also been suggested to elicit GABA currents in hippocampal neurons (Elsas et al., 2010).

The flavonoids content of P. incarnate exerted the modulation of the GABA system, e.g. chrysin and/or homoorientin, orientin, vitexin, and isovitexin (Mani and Natesan 2018; Mark Jr et al., 2008; Ropper 2005). In addition, Borges et al. (2016) and Souza et al. (2015) confirmed that chrysin flavonoids can improve the brain (hippocampal) dysfunction and reduce depressive behavior via elevation of brain-derived neurotrophic factor (BDNF). Furthermore, apigenin, the main flavonoid of P. quadragularis extract, can enhance GABAergic system, leading to a sedative activity (Gazola et al., 2015). In addition, Gazola et al. (2018) results showed that the GABAA/benzodiazepine receptors are involved in the sedative activity of vitexin-2″-O-xyloside (V2OX), the main flavonoid of P. quadrangularis’ extract.

Our results revealed a significant decrease in noradrenaline and serotonin in the epileptic brain, which came in parallel with the findings of other studies, e.g. Madhyastha et al. (2005). In addition, Singh et al. (2012) recorded a retention of serotonin and noradrenaline levels of the brain after treatment with P. incarnata. They referred such retention to the presence of alkaloids contents, e.g. harmala alkaloids. These alkaloids are reversible monoamine oxidase (MAO)-A inhibitor, thereby prevent the degradation of serotonin and noradrenaline (Abourashed et al., 2003). Therefore, the recorded ameliorative increased levels of serotonin and noradrenaline in the present study might be due to MAO-A inhibitory action of harmala alkaloids content in P. incarnata. In conclusion, the bioactive metabolites of P. incarnata might ameliorate the different consequences of epilepsy.

The inflammatory response that occurs in brain tissues during epilepsy involves MHC class I and II proteins, and macrophages activation, and CD4⁺ and CD8⁺ lymphocytes (Atkinson and Eisenbarth, 2001). The cytokine is a biomarker for inflammation and predict the next seizure episodes (Wang et al., 2015). The present data revealed a significant elevation for different proinflammatory cytokines e.g. IL-1, Il-6, TNF-α and IL-17 in epileptic non-treated group. These findings came in parallel with Wang et al. (2015) and Huang and Zhu (2018). Some studies reported that the level of serum IL-1β, IL-6 and IL-17 were significantly elevated in CSF patients with epilepsy (Mao et al., 2013; Peltola et al., 2000; Pernhorst et al., 2013; Sinha et al., 2008; Uludag et al., 2013). Other studies reported that after injury and breakdown of blood–brain barriers (BBB), elevated TGF-β expression resulted from the activated astrocytes and microglia. These events came in correlation with the neural dysfunction resulting in elevation of IL-1, IL-6, IL-8, IL-10, G-CSF, TNFα and MCP-1 (Ajmo Jr et al., 2008; Das et al., 2012; Morganti-Kossmann et al., 2010; Walker et al., 2010).

Regarding IL-6, it was elevated after focal and generalized seizures in epileptic patients (Alapirtti et al., 2009; Bauer et al., 2009; Lehtimäki et al., 2004; Lehtimäki et al., 2007). It was similarly increased in many neurological diseases such as trauma, Alzheimer’s disease and meningitis (Bauer et al., 2009; Frei et al., 1988; Woodroofe et al., 1991). Wang et al. (2015) reported that IL-6 was a unique cytokine associated with seizure severity. Therefore, IL-17 (Mao et al., 2013) and IL-6 (Wang et al., 2015) may be proposed as candidates to indicate seizure severity in general.

The present data came in parallel with others studies, e.g. Dinarello (2011); Choi et al. (2011); Zurolo et al. (2011) and Wang et al. (2015). The maximal increase in proinflammatory cytokines was reaching a high peak after six hours; IL-1 (445%), IL-6 (405%), and TNF- α (264%). They confirmed such high fold increment in glial cells within areas of intense microglia activation. The elevated proinflammatory cytokines may be due to the activated microglia cells, astrocytes, and cytokine production in late phases after seizures.

Strongly correlated with the recorded level of GABA, Prud'homme et al. (2013) reported that GABA suppresses both T cells and macrophages; suppresses production of the inflammatory cytokines; and increases TGF-beta production and regulatory T cells (Treg; IL-17). The recorded decreased level of GABA in epileptic group came in parallel with the significant high levels of IL-1β, IL-6, TNF-α and Il-17, released from the activated astrocytes and microglia (Su et al., 2015; Wang et al., 2000). Taken together, these events might contribute to the neuronal excitability via inhibition of the GABA system during epileptogenesis.

The present data revealed a strong correlation between neurotransmitters and oxidative stress imbalance and the pro-inflammatory cytokines levels in epilepsy group. The present data demonstrated that treatment with P. incarnata significantly mitigates the neuroinflammation events. In this context, chrysin as a monoflavonide of P. incranata is known as a potent antioxidant and anti-inflammatory agent Lee and Park 2015). In addition, chrysin could be a potential prophylactic agent for cerebral ischemia/reperfusion injury (Yao et al., 2014). Mani and Natesan’s (2018) study confirmed that chrysin prevents cellular membrane damage, protein damage, and the imbalance of cellular functions, via limiting the lipid peroxidation. They added that chrysin could bind to COX-2 and limit inflammatory cytokines via blocking NF-kB activation. Chrysin also significantly suppresses the serum levels of pro-inflammatory cytokines, IL-1β, IL-6, TNF-α and TGF-β expression and inhibits NF-кB activation (Ahad et al., 2014). Moreover, chrysin is reported as an inhibitory agent for TNF-α and IL-1β (Bai et al., 2013). The anti-inflammatory activity of chrysin was reported via blocking histamine release and pro-inflammatory cytokine expression (Bae et al., 2011). Therefore, Bae et al. (2011) confirmed a molecular basis for the neuroprotective effects of chrysin in ischemia/reperfusion injury.

Furthermore, the present results recorded a significantly high level of TNF-α in sera of epileptic untreated rats. TNF-α induces the recruitment of inflammatory cells and promotes ROS generation. Therefore, chrysin flavonoid contents may significantly reduce the expression of TNF-α in epileptic treated rats. This finding demonstrates the previously-reported efficacy of chrysin as a potent inhibitor of the TNF-α pathway (Lee and Park, 2015).

However, the present data showed more efficiency of P. incarnata to decrease CD4⁺ and CD8⁺ activities. These differences on T lymphocytes proliferation may be related to the different types of the phenolic compounds content (Atoui et al., 2005; Colomeu et al., 2017; Zucolotto et al., 2012). In addition, the anti-inflammatory activities of P. incarnata may be related to their polyphenols contents, e.g. Rutin, Isoorientin, Vitexin, and Catechin; due to their capability to decrease the inflammatory cells by anti-proliferative action (Colomeu et al., 2017; Tang et al., 2014).

In conclusion, the anticonvulsant effects of P. incarnata may be due to its antioxidant and anti-inflammatory actions, as well as reduction in IL-1β; Il-6, IL-17, TNF-α and TGF-β levels and inhibition in amino acids and GABAergic. However, unlike other natural products, the therapeutic benefits of P. incarnata remain nascent in current literature. Taken together, the present work tries to support further attempts to modulate seizures and epilepsy manifestation by means of modulating the inflammatory cascades using P. incarnata.

7 Availability of data and materials

The data used to support the findings of this study are included in the article.