Metabolomic Profiling and In Vivo Antiepileptic Effect of Zygophyllum album Aerial Parts and Roots Crude Extracts against Pentylenetetrazole-Induced Kindling in Mice

Abstract

The chemical profiles of both Zygophyllum album (Z. album) aerial parts and roots extracts were evaluated with LC-ESI-TOF-MS/MS analysis. Twenty-four compounds were detected. Among them, some are detected in both the aerial parts and the roots extracts, and others were detected in the aerial parts only. The detected compounds were mainly flavonoids, phenolic compounds, triterpenes and other miscellaneous compounds. Such compounds contribute to the diverse pharmacological activities elicited by the Z. album species. This study aimed to elucidate the antiepileptic effect of Z. album aerial parts and roots crude extracts against pentylenetetrazole (PTZ)-induced kindling in mice. Male albino mice were divided into four groups, eight animals each. All groups, except the control group, were kindled with PTZ (35 mg/kg i.p.), once every alternate day for a total of 15 injections. One group was left untreated (PTZ group). The remaining two groups were treated prior to PTZ injection with either Z. album aerial parts or roots crude extract (400 mg/kg, orally). Pretreatment with either extract significantly reduced the seizure scores, partially reversed the histological changes in the cerebral cortex and exerted antioxidant/anti-inflammatory efficacy evinced by elevated hippocampal total antioxidant capacity and SOD and catalase activities, parallel to the decrement in MDA content, iNOS activity and the TXNIB/NLRP3 axis with a subsequent decrease in caspase 1 activation and a release of IL-1β and IL-18. Moreover, both Z. album extracts suppressed neuronal apoptosis via upregulating Bcl-2 expression and downregulating that of Bax, indicating their neuroprotective and antiepileptic potential. Importantly, the aerial parts extract elicited much more antiepileptic potential than the roots extract did.

Keywords: LC-ESI-TOF-MS/MS; NLRP3 inflammasome; PTZ; Zygophyllum album; apoptosis; metabolomic profiling; oxidative stress.

Figure 1

Metabolites identified in Zygophyllum album crude aerial and roots extracts using LC-ESI/TOF/MS/MS.

Figure 2

Induction of kindling was achieved following fifteen PTZ injections (35 mg/Kg, i.p., three times a week for five weeks) with the seizure scores being assessed according to the Fischer and Kittner scoring scale. The effect of Z. album aerial parts and roots crude extracts on (A) mean seizure scores over the fifteen PTZ injections, (B) the AUC and (C) the final seizure score of the experimental mice. PTZ = pentylenetetrazole and AUC = area under the curve. Data are expressed as mean ± SD. Analysis was performed using one-way ANOVA followed by Tukey’s post hoc test (n = 8). ^$ significantly different vs. the normal control group; * significantly different vs. the PTZ group; ^a significantly different vs. the aerial extract (400 mg/kg) group. Differences were considered significant at p < 0.01.

Figure 3

The effect of Z. album aerial parts and roots crude extracts on the levels of (A) iNOS, (B) MDA, (C) TAC, (D) SOD and (E) catalase. iNOS = inducible nitric oxide synthase; MDA = malondialdehyde and TAC = total antioxidant capacity. Data are expressed as mean ± SD. Analysis was performed using one-way ANOVA followed by Tukey’s post hoc test (n = 8). ^$ significantly different vs. the normal control group; * significantly different vs. the PTZ group; ^a significantly different vs. the aerial extract (400 mg/kg) group. Differences were considered significant at p < 0.01.

Figure 4

The effect of Z. album aerial parts and roots crude extracts on the relative gene expression of (A) NLRP3, (B) TXNIP and (C) caspase-1 and levels of (D) IL-1β and (E) IL-18. NLRP3 = nucleotide oligomerization domain-like receptor protein 3; TXNIP = Thioredoxin-interacting protein; IL-1β = interleukin-1 beta and IL-18 = interleukin-18. Data are expressed as mean ± SD. Analysis was performed using one-way ANOVA followed by Tukey’s post hoc test (n = 8). ^$ significantly different vs. the normal control group; * significantly different vs. the PTZ group; ^a significantly different vs. the aerial extract (400 mg/kg) group. Differences were considered significant at p < 0.001.

Figure 5

Histopathological stained brain sections. Normal group showed uniform neuronal tissue with many viable uniform neurons (red arrowheads). In the PTZ group, few viable neurons are seen (red arrowheads) with many red neurons (black arrows), perineuronal edema (black arrowheads) and gliosis (red arrows), as well as many lymphocytic infiltrates (blue arrows) being observed. The aerial extract group showed a substantial increase in the number of viable neurons (red arrowheads), yet decreased red neurons (black arrows), attenuated perineuronal edema (black arrowheads), gliosis (red arrows) and lymphocytic infiltrates (blue arrows). The root extract group showed a mild increase in the number of viable neurons (red arrowheads). There are some red neurons (black arrows) and moderate perineuronal edema (black arrowheads), gliosis (red arrows) and lymphocytic infiltrates (blue arrows). (H&E, 200×. Scale bar = 50 µm).

Figure 6

The effect of Z. album aerial parts and roots crude extracts on the immunoexpression levels of apoptotic markers in the brain tissue of the experimental mice. (A) Bcl-2 expression and (B) Bax expression. Positive immunofluorescence cytoplasmic reactions are indicated by black arrows. Bcl-2 = B cell lymphoma-2 and Bax = Bcl-2 associated x. Data of the percentage of positive stained area are expressed as mean ± SD. Analysis was performed using one-way ANOVA followed by Tukey’s post hoc test (n = 8). ^$ significantly different vs. the normal control group and * significantly different vs. the PTZ group. Differences were considered significant at p < 0.05. (IHC, 200×. Scale bar = 50 µm).

Figure 6

The effect of Z. album aerial parts and roots crude extracts on the immunoexpression levels of apoptotic markers in the brain tissue of the experimental mice. (A) Bcl-2 expression and (B) Bax expression. Positive immunofluorescence cytoplasmic reactions are indicated by black arrows. Bcl-2 = B cell lymphoma-2 and Bax = Bcl-2 associated x. Data of the percentage of positive stained area are expressed as mean ± SD. Analysis was performed using one-way ANOVA followed by Tukey’s post hoc test (n = 8). ^$ significantly different vs. the normal control group and * significantly different vs. the PTZ group. Differences were considered significant at p < 0.05. (IHC, 200×. Scale bar = 50 µm).