ABSTRACT

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.

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.

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).

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.

INTRODUCTION

Epilepsy is neurological and seems to be systemic disorder (Yuen et al.¹). Epilepsy presents as partial or generalized seizures along with psychiatric comorbidities (Banerjee et al.²). Furthermore, people suffering epilepsy are more represented in the middle and low-income countries due to the enormous treatment gaps. Social stigma, discrimination and misunderstanding are some prejudices to live with epilepsy (Moshé et al.³). In the recent development of various antiseizure drugs (AEDs), some prevents seizures by acting through multiple ways such as acting on sodium channels (valproic acid, phenytoin, carbamazepine), calcium channels (ethosuximide), α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (parampanel), γ-aminobutyric acid (GABA) receptors (diazepam), or modulate the release of GABA (gabapentin, pregabalin or valproic acid) (Macdonald⁴, Kobayashi et al.⁵). Unfortunately, some of them still exhibit adverse effects like hypersensitivity reactions, mood changes, hepatotoxicity and are also ineffective against drug-resistant seizures (Copmans et al.⁶, Devinsky et al.⁷). Whereas, the primary goal of AED therapy is complete freedom from seizures without adverse side effects (Shorvon et al.⁸). So, the needs to develop new AEDs become imperative (Moshé et al.³). Pentylenetetrazol (PTZ) causes seizures on its administration by preventing inhibitory effects of GABA type A (GABA_A) receptor (Huang et al.⁹). Thus, this GABA receptor antagonist is widely used as a model for AED research. Natural sources like plants are suitable for development of various new drug candidates (Cragg et al.¹⁰).

Mitragyna inermis (Willd) Otto Kuntze (Rubiaceae) is a bushy tree or shrub (Arbonnier¹¹). In Burkina Faso, its leaves, roots and stem barks are used in mental illness and epilepsy treatment (Arbonnier¹¹, Nacoulma¹²). Different modes of preparation such as maceration, infusion, decoction and leaching with various accessible solvents such as water, hydroalcoholic and acetonic solutions are used to obtain traditional medicines (Ouédraogo et al.¹³).

Moreover, previous studies were demonstrated anticonvulsant properties of leaf methanolic extract (300-1200 mg/kg) and stem bark ethanolic and aqueous extracts (62.5-500 mg/kg) (Timothy et al.¹⁴, Atinga¹⁵). Those studies were reported on a moderate dose of convulsant agent (PTZ 60 mg/kg) and the anticonvulsant extract constituents were not specifically reported from this plant (Atinga et al.¹⁵). Therefore, these authors reported flavonoids, tannins, alkaloids, anthraquinone, glycosides, and terpenoids in these extracts. However, tube tests reported on these potent extracts are preliminary phytochemical screenings and can be confirmed or refuted by thin layer chromatography screenings (Ouédraogo et al.¹³). Also, extensive phytochemical report revealed that M. inermis contains polyphenols, triterpenoids, indole and oxindole alkaloids (Shellard and Sarpong^16,17, Toklo et al.¹⁸, Ouédraogo et al.¹⁹). It has been reported that flavonoids and alkaloids or medicinal plant containing flavonoids and alkaloids interact with GABA_A receptors leading to anticonvulsant activities (Copmans et al.⁶, Jäger and Saaby²⁰, Zhu et al.²¹). Besides, several AEDs like diazepam, carbamazepine, lorazepam, midazolam, brivaracetam, piracetam, aniracetam, oxiracetam, pramiracetam, nefiracetam, nebracetam, fasoracetam and levetiracetam licensed or in clinical development have the common function like alkaloids (Shorvon et al.⁸). Nevertheless, the knowledge of the potential contribution of flavonoids and alkaloids patterns of M. inermis different part extracts on its therapeutic properties is unknown. In drug research based on traditional recipes, it is important to assay or highlight ubiquitous tracers of biological activity due to synergistic effects (Yuan et al.²²). Indeed, flavonoids are generally followed in the standardization of raw materials from traditional medicine and chromatographic fingerprinting can be used in the quality control of medicinal plant materials (Ouédraogo et al.¹³).

This study aimed to investigate whether M. inermis organic and aqueous extracts are able to control seizures induced by PTZ in mice based on flavonoid fingerprints and alkaloidal contents.

MATERIALS AND METHODS

RESULTS AND DISCUSSION

AED are more or less effective against generalized, myoclonic and absent tonic-colonial seizures (Macdonald⁴). Indeed, around 30% of patients do not respond to the currently available AEDs (Moshé et al.³). In traditional medicine, M. inermis is used to manage epilepsy. Unfortunately, there was no data on the required levels of herbal materials for therapeutic value. Chromatographic fingerprinting is used in the quality control of medicinal plant materials (Ouédraogo et al.¹³). This report focused first on flavonoid fingerprints as tracers of bioactive contain in various fractions of M. inermis. As a result, DCM and hexane fraction didn’t show the remarkable flavonoid fingerprints (Figures 2, 3, 4). In the different parts, decoction, acetone, ethyl acetate and butanol fractions revealed various flavonoid fingerprints. Using flavonoid fingerprints, as fraction standardization, aqueous decoctate of leaves appeared atypical however, leaves ethyl acetate, butanol and acetone fractions appeared typical. Also, stem bark ethyl acetate and acetone fractions appeared typical, as well as root barks ethyl acetate and acetone fractions. Thus, for the same part, fractions may be combined for their similar flavonoid fingerprints. We noticed variability in flavonoids groups contain (Figures 2, 3, 4). Other studies reported the presence of flavonoids in M. inermis parts but still on content analyses (Nacoulma¹², Timothy et al.¹⁴, Atinga¹⁵, Ouédraogo et al.³³).

Secondly, we conducted an investigation of M. inermis roots, leaves, and stem bark standardized fraction effects on acute PTZ-induced seizures in NMRI mice. All findings were presented in Table 1 and Figure 5. The preliminary study revealed that leaf decoction, stem bark ethyl acetate extract and ethanolic extract delayed the onset of PTZ-induced convulsions. In contrast, root fractions did not show antiseizure effects in the PTZ-induced seizure test in mice (Table 1). The using of M. inermis root in the management of epilepsy has not been reported in any ethnobotanical survey. This lack of antiseizure properties might justify its non-utility in traditional medicine. Herbal medicinal product must be manufactured from the indicated part of the plant and must be free of other parts of the same or other plants (Ouédraogo et al.¹³). The acetone stem bark fraction failed to demonstrated any effect, but the ethyl acetate stem bark fraction was found to be responsible of the combined antiseizure effects (Table 1). With the aim to confirm the abilities of M. inermis-derived fractions to reduce the seizures, more assays were carried out (Figure 5). PTZ 110 mg/kg bw showed FMJ at 49 ± 0.67 sec and HLT at 65.33 ± 4.22 sec in control group. So, ethyl acetate extract at 500 mg/kg bw, decoction at 300 mg/kg bw and ethanol extract at 500 mg/kg bw revealed the noticeable delay of HLTE occurs (more than 10 min). Moreover, no significant antiseizure effect was observed between stem bark ethanolic extract (500 mg/kg bw) and leaf decoction (300 mg/kg bw) (p ˃ 0.05). In contrast, all the fractions failed to significantly delay the onset of first myoclonic seizures. These results suggested that M. inermis delay the seizures, specifically delayed HLTE occurred, but failed to show the abilities to delay first myoclonic seizures. The lack of abilities to delay the onset of FMJs suggests that M. inermis might require more time to exhibit antiseizures effects after the administration of the extract. However, the results observed in this study with the stem bark ethanolic extract are in agreement with the reported on temporary absence of seizures at 500 and 250 mg/kg bw (Timothy et al.¹⁴). Also, we reported that M. inermis leaves methanolic extract prevented seizures at the doses more than 600 mg/kg (Atinga¹⁵). But this latter study employed high doses of extracts. In traditional drug preparation methanol is subjected to usage limits (Ouédraogo et al.¹³). The high dose of PTZ used in this study to induced seizure may justify variability of anticonvulsant properties and lack of remarkable latency to FMJ occurred compared to the reported study (Timothy et al.¹⁴, Atinga¹⁵). It has been reported that flavonoids or medicinal plant extracts containing flavonoids interact with GABA_A receptors leading to anticonvulsant activities (Copmans et al.⁶, Jäger and Saaby²⁰, Zhu et al.²¹). Indeed, the variability could arise from the difference of flavonoid fingerprints observed with leaves and stem bark leading to the variation of the HLTE delay. Moreover, it may be suggested that these active extracts might act as agonist of GABA_A receptor, similar to the effects other AEDs that prevent seizures in PTZ-induced seizure test (Kobayashi et al.⁵, Shorvon et al.⁸,). So those derived fractions could be powerful sources of anticonvulsants. In addition, multiple studies reported lower toxicity of M. inermis different part-derived extracts in mice and rates (Timothy et al.¹⁴, Monjanel-Mouterde et al.³⁴, Ouedraogo et al.³⁵). Our latest research on these mixed fractions revealed that they are less hazardous to mouse fibroblast and human hepatocyte cell lines (Ouédraogo et al.¹⁹). Flavonoids could therefore be tracers to guarantee the quality and therapeutic value of M. inermis parts preparation in traditional use and the research of anticonvulsant phytomedicines.

A thorough phytochemical analysis was carried out on the most anticonvulsant extracts and stem bark ethanolic extract and leaf decostate. The fractionation revealed that the alkaloid fraction represented 2.4% of leaf decoction and 7.8% of stem bark ethanolic extract. Traore et al.³⁶ reported that alkaloid-rich fraction showed low toxicity on monocyte proliferation through inhibition of mammary cell protein production but no mutagenic or genotoxic activities. Subsequently, in order to know these fraction alkaloid ingredients, TLC on the derived alkaloid fractions offered the main oxindole alkaloids viz pteropodine, isopteropodine and mitraphylline. In addition, we isolated the new tetracyclic oxindol alkaloid, corynoxeine (Figure 6). Moreover, pteropodine and isopteropodine were reported for their lack of adverse effects on cell culture and DNA (Lee et al.³⁷, Aponte et al.³⁸, Ahmad and Salim³⁹). Also, isopteropodine showed a neuroprotective effect without toxicity until 10³ µM (Ahmad and Salim³⁹). However, M. inermis alkaloid content was already reported (Shellard and Sarpong^16,17, Toure et al.⁴⁰). Alkaloid extract and compounds of other Mitragyna species as well as M. speciosa are reported to exhibit benefit pharmacological effects on the brain, but require moderate doses (Nelsen et al.⁴¹). Indeed, alkaloid such as mitragynine interact with neurons by blocking calcium channel (Suhaimi et al.⁴²). Therefore, neuronal Ca²⁺ channel-blocking effect abolish seizures (Kobayashi et al.⁵). Mitragynine is also reported in M. inermis leaf and stem bark (Shellard and Sarpong¹⁶, Toure et al.⁴⁰). However, indole alkaloids such as ibogaine similarly to pteropodine, isopteropodine and mitraphylline isolated in this study is reported to exhibit anticonvulsant activity by blocking n-methyl-D-aspartate (NMDA) receptors. Also, tetracyclic oxindole alkaloids such as rhynchophylline and isorhynchophylline present in M. inermis leave and stem bark have been reported with anticonvulsant activities by inhibiting NMDA (Zhu et al.²¹). Thus, the abilities of decoction and ethanolic extracts to exhibit acute antiseizure effects in PTZ-induced seizure test might be attributed to the alkaloid fractions with the plausible synergistic action of pteropodine, isopteropodine, corynoxeine and mitraphylline or due to flavonoid contains. Some AEDs such as phenytoin, carbamazepine and diazepam exhibit alkaloid functions (Kobayashi et al.⁵). However, the pro-seizure effect reported with the ingestion of M. speciosa alkaloids calls for having moderate look and use of M. inermis alkaloids for treating seizures.

CONCLUSION

This study suggests that flavonoid fingerprints are tracers of M. inermis anticonvulsant ingredients. Stem bark ethanolic and ethyl acetate extracts and leaf aqueous extracts contain anticonvulsant bioactive principles that delay HLTE in male NMRI mice. Furthermore, alkaloid contents also remain plausible bioactive anticonvulsant principles. All observations support the traditional use of M. inermis to manage epilepsy. However, further studies might help understand the effects of alkaloid fractions, flavonoids, and the isolated compounds as promising antiseizure agents derived from M. inermis in experimental animals.

Phytochemical study

Plant materials and extract preparation

Based on a previous study, M. inermis parts (leaf, stem bark and root) samples were collected from Banfora, Dindérésso, and Boromo during September and November (Ouédraogo et al.²³). A voucher specimen (UNB 939) is deposited in the Herbarium of University Nazi Boni. The collected samples were subjected to total phenolic compounds assays and antioxidant properties evaluation, which are sensitive to ecological factors (Ouédraogo et al.²³). At the end of these analyses, three samples were categorized by plant part. Based on their phenolic contents and remarkable antioxidant potential, three samples by plant part were used to form composite samples per part, i.e. 40 + 40 + 40 = 120 g of stem bark, 40 + 40 + 40 = 120 g of root bark, and 30 + 30 + 30 = 90 g of leaves. The composite samples were used for different extractions, maintaining the ratio of plant matter. Different preparations have been made in relation to the traditional way of preparation and those reported from previous studies (Timothy et al.¹⁴; Ouédraogo et al.¹⁹).

Acetonic extraction

Ninety g of leaves, 120 g of root bark, and 120 g of stem bark were subjected to 10 fold 70% acetone (v/w) for 1 hour 30 min at 1500 rpm at 37 °C. Then, filtration and centrifugation at 3800 rpm/35 min/4 °C were carried out. The supernatant solvent were evaporated at 45 °C with rotary evaporator and stored at 4 °C until their use (Checkouri et al.²⁴). This extraction yielded 32.74%, 13.76%, 22.87% from leaf, stem and root, respectively.

Aqueous decoction and fractionation

Five hundred g of each part of M. inermis was used for extraction. Thus, the extraction was performed with 10 fold distilled water (v/w) 100 °C/30 min (Ranilla et al.²⁵). Filtrate pH was reduced to 3-4 and then subjected to fractionation successively with increasing polarity [hexane, dichloromethane (DCM), ethyl acetate to butanol]. The residual water and butanol were evaporated by freeze-drying and the other solvents at 45 °C using a rotary evaporator. This fractionation yielded 13.6%, 5.97%, and 4% for the leaf decoction, ethyl acetate, and butanol fractions, respectively. Also, 2% for the ethyl acetate fraction of stem bark and 3.10% for the ethyl acetate fraction of roots. Extracts and fractions were stored at 4 °C until use (Silva et al.²⁶).

Ethanol extraction

Stem bark powder (120 g) was macerated for 24 hours with 1200 mL of ethanol (99%). Therefore, the raw material was macerated twice (the second with the recovered solvent). The solvent was then evaporated at 45 °C with a rotary evaporator (2% yield) and stored at 4 °C (Timothy et al.¹⁴).

Flavonoid fingerprinting, bioassay-guided fractionation, and alkaloids isolation

Extracts and fractions of each plant part were dissolved in methanol and applied on F254 silica plate. Then, elution was carried out over 8 cm with ethyl acetate: acetic acid: methanol: water (10:1.6:0.6:1.5) and the plates were viewed at ultraviolet (UV) 365 nm and after spraying with aluminum chloride (Ouédraogo et al.¹⁹). This first phytochemical screening is part of the confirmation of the presence of different subgroups of flavonoids reported as potential bioactive of traditional drugs. In addition, this screening allows the grouping of fractions according to the tracers that are flavonoids in a preliminary standardization guide. All extracts were subjected to anti-PTZ-induced seizures assays.

Furthermore, 20 g of the aqueous decoction of the leaves and 10 g of the ethanolic extract of the stem barks that exhibited potent antiseizure effects in PTZ-induced seizures were used for alkaloid treatments. Therefore, the extracts were treated with 10% acetic acid for 12 hours. At the end of the time, filtration was performed and the pH of the filtrate was adjusted to 10, followed by extraction with DCM three times. The DCM fraction was then concentrated in rotavapor and dissolved in 5% sulfuric acid, followed by extraction with hexane to remove non-alkaloids. The residual aqueous phase pH was increased to 10 using ammonia, followed by a new extraction with DCM. The last DCM fraction was concentrated in rotavapor, dissolved in methanol, and dried under the hood. Finally, DCM was used to recover the yellow-colored-soluble compounds. This process yielded 2.4% alkaloid fraction from the leaves and 7.8% alkaloid fraction from the stem bark. Similar spots were observed on both fractions after application on a precoated F254 silica plate and elution with DCM: acetone (9:1). Then, preparative thin layer chromatography (TLC) with the same system yielded four compounds: 1 (10 mg) and 2 (10 mg) from the leaves and 3 (6 mg) and 4 (5 mg) from the stem bark.

General methods

TLCs were performed on preheated Kieselgel 60 F254 (Merck) plates. Plates were developed using DCM-acetone and then UV 254 and 365 nm. The isolated compounds were subjected to ¹H NMR, 2D NMR, HMBC, HSQC, COSY and NOESY, ¹³C NMR, and FAB-MS (+ve or –ve) analyses. Bruker Avance Neo NMR spectrometers operating at 400 and 600 MHz were used to run 1D and 2D NMR spectra, giving coupling constants (J) in Hz and chemical shifts (δ) in ppm relative to the residual CD₃OD signal with TopSpin.

Structure elucidation of pteropodine (1), isopteropodine (2), mitraphylline (3), and corynoxeine (4)

Elucidation was carried out by comparing ¹H NMR, 2D NMR, ¹³C NMR, and FAB-HR (+ve or –ve) data with the reported data.

Pteropodine (1)

Amorphous white powder: FAB-HR (+ve): m/z= 369.1826 [M + H]⁺; [M (C₂₁H₂₄O₄N₂) + H]= 369.1826. ¹H NMR (400 MHz, CD₃OD): δ = 8.53 (brs, NH), 2.34 (dd, J= 6.0, 2.4 Hz, H-3), 3.21 (dd, J= 8.2, 7.0 Hz, H-5a) 2.40 (t, J= 4.3 Hz, H-5b), 2.29 (dd, J= 3.2, 8.2 Hz, H-6a) 2.00 (ddd, J= 12.9, 7.9, 1.5 Hz, H-6b), 7.05 (td, J= 7.6, 1.1 Hz, H-9), 6.86 (d, J= 7.9 Hz, H-10), 7.19 (td, J= 7.7, 1.3 Hz, H-11), 7.27 (dd, J= 7.5, 1.2 Hz, H-12), 1.55 (d, J= 7.6 Hz, H-14a) 1.33 (dd, J= 3.7, 7.2 Hz, H-14b), 2.37 (dd, J= 3.3, 2.5 Hz, H-15), 7.50 (s, H-17); 4.47 (dq, J=12.4, 6.1 Hz, H-19), 1.59 (d, J= 4.6 Hz , H-20), 2.32 (d, J= 2.3 Hz, H-21a), 3.31 (H-21b), 3.57 (s, 3H, OCH₃), 1.39 (d, J= 6.1 Hz, CH₃). ¹³C NMR (600 MHz, CD₃OD): δ = 183.36 (C-2), 75.31 (C-3), 55.91 (C-5), 34.98 (C-6), 57.76 (C-7), 134.60 (C-8), 123.79 (C-9), 110.57 (C-10), 129.19 (C-11), 124.18 (C-12), 142.78 (C-13), 30.84 (C-14), 32.31 (C-15), 110.57 (C-16), 156.83 (C-17), 73.72 (C-19), 39.29 (C-20), 54.27 (C-21), 51.67 (OCH₃), 169.66 (C=O), 18.95 (CH₃).

Infrared spectroscopy (IR) [potassium bromide (KBr)]: V_max= 3320.57, 2943.98, 2832.23, 668.42 cm^-1. UV/UV-visible (MeOH) A= 1.347 (218 nm) and 1.652 (247 nm).

Isopteropodine (2)

Amorphous white powder: FAB-HR (+ve): m/z= 369.1821 [M + H]⁺; [M (C₂₁H₂₄O₄N₂) + H]= 369.1821. ¹H NMR (400 MHz, CD₃OD): δ = 8.54 (brs, NH), 2.42 (dd, J= 4.2, 2.8 Hz, H-3), 3.25 (td, J= 8.7, 2.7 Hz, H-5), 2.29 (ddd, J= 12.4, 9.6, 2.5 Hz, H-6b) 1.96 (dt, J= 13.0, 8.5 Hz, H-6b), 7.01 (td, J= 7.6, 1.1 Hz, H-9), 6.88 (d, J= 7.7 Hz, H-10), 7.18 (td, J= 7.7, 1.3 Hz, H-11), 7.27 (d, J= 6.7 Hz, H-12), 1.47 (dt, J= 13.1, 3.8 Hz, H-14a) 0.79 (q, J= 12.2 Hz, H-14b), 2.44 (dd, J= 8.2, 3.7 Hz, H-15), 7.42 (s, H-17), 4.32 (dq, J= 10.4, 6.2 Hz, H-19), 1.59 (ddd, J= 8.8, 7.7, 4.4, 3.6 Hz, H-20), 2.39 (dd, 3.6, 8.5 Hz, H-21), 3.36 (dd, J= 12.1, 2.1 Hz, H-21b), 3.58 (s, 3H, OCH₃), 1.40 (d, J= 6.24 Hz, CH₃). ¹³C NMR (600 MHz, CD₃OD): δ = 183.17 (C-2), 72.77 (C-3), 55.10 (C-5), 35.36 (C-6), 58.51 (C-7), 135.09 (C-8), 123.43 (C-9), 110.83 (C-10), 128.98 (C-11), 125.47 (C-12), 142.35 (C-13), 31.41 (C-14), 31.92 (C-15), 110.97 (C-16), 156.50 (C-17), 73.64 (C-19), 39.36 (C-20), 54.50 (C-21), 51.56 (OCH₃), 169.40 (C=O), 18.78 (CH₃).

IR (KBr): V_max= 3320.43, 2944.11, 2832.23 cm^-1. UV/UV-Visible (MeOH) A= 0.790 (214 nm), 0.815 (244 nm).

Mitraphylline (3)

Amorphous white powder: FAB-HR (+ve): m/z= 369.1809 [M + H]⁺; [M (C₂₁H₂₄O₄N₂) + H]= 369.1809. ¹H NMR (400 MHz, CD₃OD): δ = 8.50 (brs, NH), 2.45 (d, J= 2.5 Hz, H-3), 3.25 (d, J= 2.5 Hz, H-5a), 2.55 (d, J= 1.6 Hz, H-5b), 2.06 (ddd, J= 13.1, 8.1, 1.4 Hz, H-6a), 2.34 (dd, J = 4.6, 1.7 Hz, H-6b), 7.06 (td, J= 7.6, 1.0 Hz, H-9), 6.88 (d, J= 7.7 Hz, H-10), 7.21 (td, J= 7.7, 1.2 Hz, H-11), 7.29 (d, J= 7.6 Hz, H-12), 2.25 (dt, J= 3.1, 3.0 Hz, H-14a), 0.99 (q, J= 11.6 Hz, H-14b), 2.09 (d, J= 1.4 Hz, H-15), 7.44 (s, H-17), 4.42 (dd, J= 6.5, 3.5 Hz, H-19), 1.92 (dd, J= 4.6, 2.3 Hz, H-20), 3.11 (d, J= 7.3 Hz, H-21), 1.90 (s; H-21), 3.55 (s, 3H, OCH₃), 1.11 (d, J= 6.6 Hz, CH₃). ¹³C NMR (600 MHz, CD₃OD): δ = 180.29 (C-2) , 75.56 (C-3), 54.97 (C-5), 35.48 (C-6), 57.07 (C-7), 134.71 (C-8), 123.73 (C-9), 110.56 (C-10), 129.23 (C-11), 124.14 (C-12), 143.17 (C-13), 30.72 (C-14), 31.56 (C-15), 108.17 (C-16), 155.63 (C-17), 75.43 (C-19), 42.91 (C-20), 54.92 (C-21), 51.39 (OCH₃), 168.91 (C=O), 15.08 (CH₃).

IR (KBr): V_max= 3323.82, 2943. 70, 2832.08, 1448.24, 1113.69 cm^-1. UV/UV-Visible (MeOH) A= 0.199 (230 nm), 0.303 (216 nm), 0.160 (243 nm), 0.104 (260 nm), 0.097 (267 nm).

Corynoxeine (4)

Amorphous white powder: FAB-HR (+ve): m/z= 383.1955 [M + H]⁺; [M (C₂₂H₂₆O₄N₂) + H]= 383.1955. ¹H NMR (400 MHz, CD₃OD): δ = 8.55 (NH), 2.39 (d, J= 2.0 Hz, H-3), 2.45 (d, J= 9.1 Hz, H-5a), 3.28 (H-5b), 2.30 (m, H-6a), 2.00 (d, J= 11.9 Hz, H-6b), 7.02 (td, J= 7.6, 1.1 Hz, H-9), 6.87 (d, J= 7.7 Hz, H-10), 7.18 (td, J= 7.7, 1.3 Hz, H-11), 7.43 (d, J= 8.8 Hz, H-12), 1.88 (H-14), 2.50 (d, J= 3.3 Hz, H-15), 7.25 (s, H-17), 5.49 (m, H-18), 4.90 (d, J= 2.1 Hz, H-19), 2.84 (d, J= 11.0 Hz, H-20), 3.17 (dt, J= 11.0, 3.6 Hz, H-21a) 1.95 (m, H-21b), 3.55 (s, OCH₃), 3.72 (s, OCH₃). ¹³C NMR (600 MHz, CD₃OD): δ = 180.26 (C-2), 73.88 (C-3), 55.02 (C-5), 36.06 (C-6), 58.12 -C-7), 135.0 (C-8), 123.45 (C-9), 110.72 (C-10), 128.86 (C-11), 125.94 (C-12), 142.3 (C-13), 24.17 (C-14), 73.37 (C-15), 112.56 (C-16), 161.48 (C-17), 140.87 (C-18), 115.97 (C-19), 43.6 (C-20), 60.05 (C-21), 51.29 (OCH₃), 61.85 (OCH₃), 170.31 (C=O).

IR (KBr): V_max= 3320.28, 2943.33, 2832.29, 1448.15, 1114.24 cm^-1. UV/UV-visible (MeOH) A= 0.987 (220 nm) and 1.058 (230 nm).

Compounds 1, 2, and 3 appeared as white amorphous powder with the respective molecular weights 368.1826, 368.1821 and 368.1809. All matched to the same calculated formula C₂₁H₂₄O₄N₂. In addition, the common fragment ions appeared at m/z= 339.3 [M-30]⁺, 325.2 [M-44]⁺, 291.2, 227.3, 164.1 [M-205]⁺. Herein, they reveal similarity to pentacyclic oxindole alkaloids. On TLC with solvent system DCM: Acetone (9:1), these compounds showed different frontal reference high (1 ˃ 2 ˃ 3). All compounds (1, 2, 3) in ¹H NMR revealed presence of one methoxy group. That means, these compounds are the stereoisomers of pentacyclic oxindole alkaloids. Therefore, configuration assignment based on the chemical shift described by Beckett et al.²⁷ showed that compound 1 and compound 2 are on β configuration with C19, C20 but α configuration with C15. Also, compound 1 (δd-CH₃= 1.39) and compound 2 (δd-CH₃= 1.40) revealed cis D/E ring junction with three-proton doublet for the methyl (Beckett et al.²⁷). Compound 3 three-proton doublet appeared at 1.11 ppm as D/E ring trans junction (Beckett et al.²⁷). Correlation was observed between H-17 doublet and the proton H-15 via a long range in compounds 1, 2 and 3. However, according to asymmetric centers and their NMR spectra assignment by theoretical calculations of shielding constants of Paradowska et al.,²⁸ compound 1 (δC3 = 75.31) and compound 3 (δC3 = 75.56) appeared as 7R alkaloids, but compound 2 (δC3 = 72.77) seemed like 7S alkaloid. Also, compound 1 (δC20 = 39.29) and compound 2 (δC3 = 39.36) revealed to be 20S alkaloid but 20R alkaloid for compound 3 (δC3 = 42.91) (Paradowska et al.²⁸). For that, compound 1 (7R, 20R) allo-type isomer is identified to pteropodine (uncarine C), compound 2 (7S, 20R) also allo-type isomer is identified to isopteropodine (uncarine E) and compound 3 (7R, 20R) normal type isomer matched to mitraphylline (Paradowska et al.²⁸, Toure et al.²⁹, García Giménez³⁰, Salim and Ahmad³¹). Full ¹H and ¹³C NMR signals assignment was made using HMBC, HSQC, COSY and NOESY correlation data. These compounds were previously reported from M. inermis.

Compound 4 appeared in molecular weight and formula 382.1955 and C₂₂H₂₆O₄N₂ respectively. Peaks at m/z= 339.2, 325.2 and 164.0 revealed the presence of olifinic and oxindole fragments. ¹H NMR revealed the presence of two methoxy groups at δH= 3.72, δC= 61.85 and δH= 3.55, δC= 51.29. Paradowska et al.²⁸ theoretical calculations of shielding constants were used for oxindole alkaloids asymmetric centers and NMR spectrum assignment. So, compound 4 (δC3 = 73.88; δC20 = 43.6) is (7R, 20R) alkaloid. The chemical shifts δC18 = 140.87, δH18 = 5.49 and δC19 = 115.97, δH19 = 4.90 are characteristic of the methylene group. It appeared to be a tetracyclic oxindol alkaloid. However, ¹H NMR, 2D NMR, ¹³C NMR and FAB-HR compound 4 matched to those of rhyncophylline 18, 19 didehydro like corynoxeine reported from M. speciosa (Paradowska et al.²⁸, Flores-Bocanegra et al.³²). Thus, in this study, we isolated new specific tetracyclic oxindole named corynoxeine from M. inermis.

Acute PTZ-induced seizure model in mice

All the procedures were performed according to the institutional animal care committee of the International Center for Chemical and Biological Sciences (ICCBS). The animal study protocol was submitted to the ICCBS/Karachi University Ethical Committee (date: 20.02.2019, approval number: 2019-006). Seventy-six male naval medical research institute (NMRI) mice with weights between 20 and 24 g were chosen for the experiment. Mice were sourced from ICCBS animal research facility. An experiment was conducted in the light of the daylight/darkness cycle at 25 ± 2 °C with appropriate moisture. Animals were brought into the experimental room 1 hour before the start of the experiment for the acclimatization and separated into control and test groups. 110 mg/kg body weight (bw) PTZ as convulsive dose was selected based on an acute crisis model. Mice were grouped together in batches of 7 mice for each dose and control. After acclimatization, the controls were treated with distilled water 10 mL/kg bw by intraperitoneal (i.p.). After 30 min, PTZ was injected into the animals, and the main stage of seizure development were observed. The extract ability to attenuate seizure threshold is indicated by absence of seizures at least 5 seconds (Atinga¹⁵). For the tests with the extracts and fractions of M. inermis, the 7 mice for each dose were subdivided into two sub-groups of 3 mice for the first test and 4 mice for the additional test. During the first phase, if no major capacity was observed to mitigate the effects of PTZ at a given dose, the additional test was not carried out.

The following doses prepared in distilled water were considered based on data in the literature (Timothy et al.¹⁴, Atinga¹⁵):

• Aqueous decoctate of leaves: 500, 300, and 150 mg/kg bw;

• Additional fractions acetone + ethyl acetate + butanol of leaves: 500 and 250 mg/kg;

• Stem bark ethanolic extract: 500 and 250 mg/kg bw;

• Additional stem bark ethyl acetate + acetone fractions: 500 and 250 mg/kg bw;

• Stem bark ethyl acetate fraction: 500 and 250 mg/kg bw;

• Stem bark acetone fraction: 500 and 250 mg/kg bw;

• Additional root bark ethyl acetate + acetone fractions: 500 and 300 mg/kg bw

• For the test groups, a suitable dose of the extract was injected intraperitoneally into the animals. After 30 min, a convulsive dose of PTZ was administered, and the delay in the onset of first myoclonic jerk (FMJ) and hind limb tonic extension (HLTE) was observed and recorded for each animal. The flowchart of the entire study is presented in Figure 1.

Statistical analysis

Statistical analysis were performed by One-Way ANOVA followed by Dunnett’s multiple comparison using Graph Pad Prism-8 Statistical Software Package at significance level set as p < 0.05.