Antiseizure Activity of Mitragyna inermis in the Pentylenetetrazol-Induced Seizure Model in Mice: Involvement of Flavonoids and Alkaloids

Abstract

Objective: This study aimed to investigate whether Mitragyna inermis (Willd.) Otto Kuntze organic and aqueous extracts are able to control seizures induced by pentylenetetrazol (PTZ) in mice based on flavonoid fingerprints and alkaloidal contents.

Materials and methods: Ethanolic extract and decoction-derived fractions from roots, leaves, and stems were subjected to chromatographic fingerprinting using AlCl₃ and screening for their antiseizure effects using PTZ-induced acute seizure model. From the fractions that showed potent bioactivities, plausible antiseizure alkaloids were isolated using thin layer chromatography, and their structures were elucidated using ¹H NMR, 2D NMR, ¹³C NMR, and FAB-HR (+ve or -ve).

Results: All fractions, with the exception of the dichloromethane and hexane fractions, revealed remarkable flavonoid fingerprints. An acute PTZ-induced seizure test revealed that ethanolic extract of stem bark [500 mg/kg body weight (bw)], ethyl acetate extract of stem bark (500 mg/kg bw), and aqueous extract of leaves (300 mg/kg bw) significantly delayed the occurrence of hind limb tonic extension (HLTE); however, a non-significant delay was observed in the onset of first myoclonic jerk compared with control animals. Isolation yielded four main alkaloids: that are, pteropodine (1), isopteropodine (2), mitraphylline (3) and corynoxeine (4). Corynoxeine is a new compound derived from M. inermis.

Conclusion: This study suggests that flavonoid fingerprints are tracers of M. inermis anticonvulsant ingredients. The stem bark ethanolic and ethyl acetate extracts and leaf aqueous extracts contain anticonvulsant bioactive principles that delay notifying the HLTE occurring in male naval medical research institute mice. Furthermore, alkaloidal contents also remain plausible bioactive anticonvulsant principles. All observations support the traditional use of M. inermis to manage epilepsy. However, further studies are needed to understand the effects of alkaloid fractions, flavonoids, and the isolated compounds as promising antiseizure agents derived from M. inermis in experimental animals.

Keywords: Mitragyna inermis; alkaloids; antiseptics; corynoxeine; flavonoids.

Figure 1

Flowchart of the entire study NMRI: Naval medical research institute, DCM: Dichloromethane, TLC: Thin layer chromatography

Figure 2

TLC of only leaves extract and fractions that contain flavonoids, A) UV light, B) AlCl3 spraying + UV light. Four additional yellow spots after spraying revealing flavonoids contain. But, EtOAc and Ac appeared as the same compound group contains TLC: Thin layer chromatography, UV: Ultraviolet, Aq: Aqueous, But: Butanol, EtOAc: Ethyl acetate, Ac: Acetone fractions

Figure 3

TLC of stem bark, A) UV light, B) AlCl3 spraying + UV light. 1) Hexane fraction, 2) DCM fraction, 3) EtOAc fraction, 4) AC. Three additional yellow spots after spraying revealing flavonoid contains. Most present in 3 and 4 that looked the same TLC: Thin layer chromatography, UV: Ultraviolet, DCM: Dichloromethane, EtOAc: Ethyl acetate, Ac: Acetone fractions

Figure 4

TLC of roots, A) UV light, B) AlCl3 spraying + UV light. 1) Hexane fraction, 2) DCM fraction, 3) EtOAc fraction, 4) Ac. Three additional yellow spots after spraying revealing flavonoid content. Most present in 3 and 4 that appeared the same TLC: Thin layer chromatography, UV: Ultraviolet, DCM: Dichloromethane, EtOAc: Ethyl acetate, Ac: Acetone fractions

Figure 5

Anti-PTZ-induced seizures of some of the most active fractions. Onsets of FMJ and HLTE are expressed as mean ± SD, n= 7. The dose 0 mg/kg bw is the control (group treated with distilled water). No significant difference was observed between the FMJ onset of the control and treated groups. Compared with the control group, stem bark ethyl acetate and ethanol extract and leave decoction delayed noticeably HLTE (p ≤ 0.05). Statistical analyses were performed by One-Way ANOVA with Dunnett’s multiple comparison tests Statistical significance levels: *p ≤ 0.05, ns: Non-significant, Dec: Decoction, EtOH: Ethanol, EtOAc: Ethyl acetate, PTZ: Pentylenetetrazol, FMJ: First myoclonic jerk, HLTE: Hind limb tonic extension, SD: Standard deviation

Figure 6

Structures of pteropodine (1), isopteropodine (2), mitraphylline (3), and corynoxeine