Introduction

Brain neuronal activities, especially neurotransmission processes are organized into systems of individually unique neuronal cells, neurotransmitters, neuromodulators, receptor molecules, and secondary messengers. Notable among these systems include the monoaminergic and cholinergic systems. Central to the cholinergic system of neurotransmission is the neurotransmitter acetylcholine. Acetylcholine (ACh) operates as a rapidly acting neurotransmitter at the neuromuscular synapses, autonomic ganglia, and the brain (Changeux 2010). ACh is hydrolyzed by a family of enzymes called cholinesterase. The prolonged inhibition of acetylcholinesterase (AChE) could ultimately lead to excessive accumulation of ACh at the synaptic cleft which will lead to overstimulation of the cholinergic neuron (Rodrigues et al. 2011). The continual overstimulation of these neurons could lead to neurodegeneration and eventual death of the organism (Nunes et al. 2003). The monoamine system of neurotransmission consists of network of neurotransmission systems, each mediated by a specific monoamine neurotransmitter. Notable among them are the dopaminergic, noradrenergic, and serotonergic systems which are mediated by dopamine, noradrenaline, and serotonin neurotransmitters, respectively. The enzyme monoamine oxidase (MAO) exists in two isoforms (MAO A and MAO B) and are both involved in the oxidative deamination of biogenic amines (neuroamines, vasoactive, and exogenous amines); thus, regulating the concentration of amine neurotransmitters as well as several amine drugs (McCabe-Sellers et al. 2006). Despite the well-reported therapeutic potentials of MAO inhibitors in the management of neurological disorders, there are several toxicological complications such as serotonin toxicity (Boyer and Shannon 2005) and hypertensive crisis (McCabe-Sellers et al. 2006) that can arise from excessive MAO inhibition which can occur as a result of excessive intake of MAO inhibitors. Furthermore, plant extracts such as from Syrian rue (Peganum harmala) (Herraiz et al. 2010) and Passion flower (Passiflora incarnata) (Dhawan et al. 2004) contain a family of effective MAO inhibitors called beta-carboline alkaloids which have records of causing adverse health effects (Santillo et al. 2014).

Jimson weed (Datura stramonium L.) is an annual herbaceous plant reported for its many pharmacological properties (Soni et al. 2012). All parts of the plant, notably the leaf, fruit, and seed are reported to be rich in alkaloids, especially the tropane alkaloids (Steenkamp et al. 2004). Despite the fact that some researchers have reported the medicinal properties of this plant (Soni et al. 2012; Altameme et al. 2015), there exist some serious neurological effects such as hallucination and anxiety, which have been reported in folklore. In addition, intentional use of the plant for its hallucinating effects has also been well documented, especially in adolescents (Adegoke and Alo 2013), while cases of Datura-induced toxicity in humans as a result of consumption of farm produce such as tea and soybean contaminated with Datura seeds have been reported (Soni et al. 2012). However, despite these serious adverse effects of this plant on psycho-activity, there is dearth of information on the effect of its phytoconstituents on critical enzymes involved in the maintenance of normal neuronal function. Consequently, this study aims to investigate the effect of crude alkaloid extracts from the leaf and fruit of Jimson weed on key enzymes of the monoaminergic (MAO) and cholinergic (AChE and BChE) systems of neurotransmission.

Materials and methods

Materials

Collection and preparation of samples

Jimson weed (D. stramonium L.) plant was collected at the stage of opening of first capsule, from local farm settlement in Akure, Ondo State (Southwest) Nigeria, during the late raining season (August) of year 2014. The plant was authenticated at the Forest Research Institute of Nigeria (FRIN), Ibadan, Oyo State (Southwest) Nigeria. A sample voucher was deposited at the institute’s herbarium (voucher number FHI 110111). Leaves and fruits of the plant were carefully separated, washed with water to remove dirt, and dried under shade for several days until a constant weight was obtained. Thereafter, the dried samples were pulverized in an electronic stainless steel blender, and stored in air-tight dark containers in the refrigerator at 4 °C for alkaloid extraction.

Chemicals and reagents

Chemicals and reagents used such as semicarbazide, benzylamine, acetylthiocholine iodide, and butyrylthiocholine iodide were procured from Sigma-Aldrich, Inc., (St. Louis, MO); trichloroacetic acid (TCA) was sourced from Sigma-Aldrich, Chemie GmbH (Steinheim, Germany); 2,4-dinitrophenyl hydrazine (DNPH) from ACROS Organics (NJ, USA); and methanol and acetic acid were sourced from BDH Chemicals Ltd., (Poole, England). All other chemicals were of analytical grade while the water used for all analysis was glass distilled.

Methods

Preparation of alkaloid extracts

Alkaloid extract of samples were prepared according to the method of Harborne (1998), with slight modifications. Briefly, 50 g pulverized sample was defatted with n-hexane for 24 h. Thereafter, 10 g of defatted samples were weighed into a 250-ml beaker and 100 ml of 10 % acetic acid in ethanol was added and covered. These were vigorously shaken, venting the mounted pressure and allowed to stand for 24 h to allow for sufficient extraction. The mixtures were thereafter, filtered first using muslin cloth and then filter paper (Whatman No. 1) to obtain a clear filtrate which was concentrated under vacuum using rotary evaporator (Laborota 4000 Efficient, Heidolph, Germany) at 45 °C. Concentrated ammonium hydroxide was subsequently added drop wise to the concentrated filtrate until the precipitate was completed. The whole solution was allowed to settle and the precipitate was collected and rinsed with dilute ammonium hydroxide to obtain the alkaloid extracts. The extracts were collected and stored in the refrigerator at 4 °C for further analysis.

Monoamine oxidase assay (in vitro)

The effect of the alkaloid extracts from the leaf and fruit of Jimson weed MAO (EC 1.4.3.4) activity was measured according to a previously reported method (Green and Haughton 1961) with slight modification. In brief, the reaction mixture contained 0.025 M phosphate buffer (pH 7.0), 0.0125 M semicarbazide, 10 mM benzylamine, 0.67 mg of the enzyme and 0–100 μl of extracts. After 30 min incubation, acetic acid was added and incubated for 3 min in boiling water bath followed by centrifugation. The resultant supernatant (1 ml) was mixed with equal volume of 2,4-DNPH, and 1.25 ml of benzene was added after 10 min incubation at room temperature. After separating the benzene layer, it was mixed with equal volume of 0.1 N NaOH. Alkaline layer was decanted and incubated at 80 °C for 10 min. The orange–yellow color developed was measured at 450 nm in a UV/visible spectrophotometer (Jenway 6305 model). The MAO activities were thereafter expressed as percentage inhibition of the reference; thus,

$$ \%\ \mathrm{inhibition}=\left[\mathrm{AB}{\mathrm{S}}_{\mathrm{ref}}-\mathrm{AB}{\mathrm{S}}_{\mathrm{sample}}\right)/\mathrm{AB}{\mathrm{S}}_{\mathrm{ref}}\Big]\times 100 $$

where ABSref = absorbance of reference and ABSsample = absorbance of sample.

Cholinesterase assay (in vitro)

The effect of the extracts on cholinesterase [acetylcholinesterase (AChE) and butyrylcholinesterase (BChE)] activities was assessed by a modified colorimetric method (Perry et al. 2000). The cholinesterase activity was determined in a reaction mixture containing 200 μl of AChE (EC 3.1.1.7) or BChE (EC 3.1.8) solution in 0.1 M phosphate buffer (pH 8.0), solution of 5,5′-dithio-bis(2-nitrobenzoic) acid (DTNB 3.3 mM), sample extracts (0–100 μl), and phosphate buffer, pH 8.0. After incubation for 20 min at 25 °C, acetylthiocholine iodide (for AChE activity assay) or butyrylthiocholine iodide (for BChE activity assay) was added as the substrate, and enzyme activity was determined in a UV/visible spectrophotometer (Jenway 6305 model) at 412 nm. The AChE and BChE activities were thereafter expressed as percentage inhibition of the reference; thus,

$$ \%\ \mathrm{inhibition}=\left[\mathrm{AB}{\mathrm{S}}_{\mathrm{ref}}-\mathrm{AB}{\mathrm{S}}_{\mathrm{sample}}\right)/\mathrm{AB}{\mathrm{S}}_{\mathrm{ref}}\Big]\times 100 $$

Where ABSref = absorbance of reference and ABSsample = absorbance of sample.

GC-MS characterization of alkaloid extracts

This analysis was performed using HP 6890 series gas chromatograph coupled with 5975C inert mass spectrometer (with triple axis detector) with electron-impact source (Agilent Technologies). The stationary phase of separation of the compounds was HP-5 capillary column coated with 5 % phenyl methyl siloxane (30 m length × 0.32 mm diameter × 0.25 μm film thickness) (Agilent Technologies). The carrier gas was helium used at constant flow of 1.6 ml/min at an initial nominal pressure of 2.84 psi and average velocity of 46 cm/s. The samples were injected in splitless mode at an injection temperature of 260 °C and injection volume of 1 μl. Purge flow was 21.5 ml/min at 0.50 min with a total flow of 25.8 ml/min; gas saver mode was switched on. An oven was initially programmed at 60 °C (1 min) then ramped at 4 °C/min to 110 °C (3 min) then 8 °C/min to 260 °C (5 min) and 10 °C/min to 300 °C (12 min). Run time was 56.25 min with a 3 min solvent delay. The mass spectrometer was operated in electron-impact ionization mode at 70 eV with ion source temperature of 230 °C, quadrupole temperature of 150 °C, and transfer line temperature of 280 °C. Scanning of possible alkaloid compounds was from m/z 30 to 550 amu at 2.62 s/scan scan rate and was identified by comparing measured mass spectral data with those in NIST 11 Mass Spectral Library and literature.

Data analysis

The results of three (3) experiments were pooled and expressed as mean ± standard deviation (SD). Mean values were appropriately analyzed and compared using Student’s t test (unpaired) and significance was accepted at P ≤ 0.05. Also, IC50 (effective concentration of extract causing 50 % inhibition) values were calculated using nonlinear regression analysis. All statistical analyses were carried out using GraphPad Prism version 5.00 for Windows.

Result

Figure 1 presents the result of the modulatory effect of the alkaloid extracts from the leaf and fruit of Jimson weed on monoamine oxidase (MAO) activity. Both extracts inhibited MAO activity in a concentration-dependent manner (0–26.67 μg/ml). Considering the IC50 value presented in Table 1, the fruit extract had a higher significant (P < 0.05) inhibitory effect (13.70 ± 0.02 μg/ml) than the leaf extract (28.64 ± 0.05 μg/ml).

Fig. 1
figure 1

Inhibitory effects (%) of alkaloid extracts from the leaf and fruit of Jimson weed (D. stramonium L.) on the activity of monoamine oxidase (MAO)

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Table 1 IC50 values for the inhibitory effects of alkaloid extracts from the leaf and fruit of Jimson weed on monoamine oxidase (MAO), acetylcholinesterase (AChE), and butyrylcholinesterase (BChE) activities
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The result of the modulatory effect of the alkaloid extracts on acetylcholinesterase (AChE) activity is presented in Fig. 2. This showed that the extracts inhibited the activity of AChE in concentration-dependent manner (0–13.04 μg/ml). However, judging by the IC50 values (Table 1), the fruit extract had a more potent (P < 0.05) inhibitory effects (5.06 ± 0.02 μg/ml) than the leaf extract (9.32 ± 0.04 μg/ml).

Fig. 2
figure 2

Inhibitory effects (%) of alkaloid extracts from the leaf and fruit of Jimson weed (D. stramonium L.) on acetylcholinesterase (AChE) activity

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Similarly, Fig. 3 presents the result of the modulatory effect of the alkaloid extracts on butyrylcholinesterase (BChE) activity. This revealed that BChE activity was also inhibited concentration dependently (0–13.04 μg/ml) by both extracts. However, as revealed by the IC50 value (Table 2), the fruit extract had more potent (P < 0.05) inhibitory effect (6.59 ± 0.02 μg/ml) than the leaf extract (14.70 ± 0.04 μg/ml).

Fig. 3
figure 3

Inhibitory effects (%) of alkaloid extracts from the leaf and fruit of Jimson weed (D. stramonium L.) on butyrylcholinesterase (BChE) activity

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Table 2 GC-MS characterization of the alkaloid extract from the leaf of Jimson weed
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The results of the GC-MS characterization of the phytoconstituents of the extracts are presented in Tables 2 and 3. The results revealed that 48 compounds were identified from the leaf extract while 56 compounds were identified from the fruit extracts. The compounds detected in the extracts include alkaloids such as atropine, scopolamine, amphetamine, 3-methoxyamphetamine, 3-ethoxyamhetamine cathine, spermine, phenylephrine, 3-piperidinemethanol, sarcosine, N-(cyclopentylcarbonyl)-, and decyl ester, among others. The percentage distribution of the compounds also shows that the alkaloids are more abundant in the fruit extract.

Table 3 GC-MS characterization of the alkaloid extract from the fruit of Jimson weed
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Discussion

Jimson weed (D. stramonium L.) is gaining popularity for its several reported medicinal and pharmacological properties, including its effect on neuromodulation. These neuromodulatory effects which include hallucination, short-term memory loss, depression, and impaired cognitive function (Adegoke and Alo 2013) have been largely linked to the alkaloid constituents of the plant (Soni et al. 2012). In this study, the observed inhibitory effects of alkaloid extracts from the leaf and fruit of Jimson weed on brain MAO activity could contribute to the mechanism by which it induces neuromodulatory properties as reported in folklore, with the fruit producing more potent MAO inhibitory effect, but was less potent than that of previously reported standard nonselective MAO inhibitor iproniazid (IC50 = 0.72 μg/ml) (Zhi et al. 2016). This finding agrees with earlier studies in which neuromodulatory properties of plant alkaloids extracts have been linked to their MAO inhibitory effects. Plant extracts such as from Syrian rue (P. harmala) (Herraiz et al. 2010) and passionflower (P. incarnata) (Dhawan et al. 2004) contain a family of effective MAO inhibitors called beta-carbolines which have records of causing adverse health effects (Santillo et al. 2014). Furthermore, Herraiz and Chaparro (2005) reported that two beta-carboline alkaloids (norharman and harman) isolated from tobacco smoke exhibited significant MAO inhibition which could be associated with cigarette-induced addiction and depression.

Despite the well-reported therapeutic potentials of MAO inhibitors in the management of neurological disorders, there are several toxicological complications that arise from excessive MAO inhibition which can occur as a result of excessive intake of MAO inhibitors, adverse drug or dietary supplement reactions, or MAO inhibitors from plant sources. For example, serotonin toxicity is a serious pathological disorder resulting from hyperactivity of serotonin neurotransmitter as a result of excessive accumulation of serotonin due to excessive MAO inhibition (Boyer and Shannon 2005). Neurostimulants such as methylenedioxymethamphetamine (ecstasy) and tetrahydrocannabinol, which are commonly used by young people for their “mind-altering” properties has been reported to induce inhibitory effects on MAO activity which may contribute to their neuromodulatory properties especially in long-term exposure (White et al. 1996; Fisar 2012).

Cholinergic toxicity occurs when there is accumulation of acetylcholine at synaptic cleft leading to overstimulation of postsynaptic neurons (Rodrigues et al. 2011). This is often due to excessive inhibition of AChE, resulting in accumulation of acetylcholine. If unchecked, cholinergic toxicity is capable of inducing severe neurological impairment, neurodegeneration, and even death of the organism (Nunes et al. 2003). Inhibition of brain AChE activity is often used as test for toxicity due to some environmental pollutants (Srivastava and Shivanandappa 2011). Therefore, in this study, the ability of alkaloid extracts from the leaf and fruit of Jimson weed to inhibit AChE activity could potentiate cholinergic toxicity and possibly neurodegeneration. Notably, the fruit extract showed a significantly higher AChE inhibitory effect compared to the leaf, but was less potent than that of previously reported standard AChE inhibitor prostigmine (IC50 = 0.046 μg/ml) (Oboh et al. 2014). Previous studies on Jimson weed have shown that the fruit and especially the seed showed higher pharmacological and neuromodulatory effects than the leaf extracts (Soni et al. 2012). Actually, the most reported cases of Datura poisoning have been attributed to voluntary or accidental ingestion of the fruit or seed. Furthermore, previous studies have reported the anticholinesterase abilities of plant alkaloid extracts (Cometa et al. 2012). Therefore, the AChE inhibitory effect of Jimson weed observed in this study can be significantly attributed to its constituent alkaloids. Hence, consumption of Jimson weed leaf and fruit either as medicinal herb for its hallucinating effects or accidentally as food contaminants could possibly induce cholinergic toxicity as a result of inhibition of AChE activity.

We also observed, in this study that both leaf and fruit alkaloid extracts from Jimson weed inhibited BChE activity with the fruit showing significantly higher inhibitory effect, but was less potent than that of previously reported standard BChE inhibitor prostigmine (IC50 = 0.044 μg/ml) (Oboh et al. 2014). Although studies have shown that AChE is predominant over BChE in healthy human brain (Giacobini 2003), and while the precise function BChE still remains to be fully understood, it is believed to be involved in regulating cell proliferation and early stage of neuronal differentiation (Mack and Robitzki 2000). In addition, BChE can also hydrolyze acetylcholine in place of AChE but with less specificity (Çokuğraş 2003); therefore, its inhibition can also lead to impaired neuronal function and neurodegeneration. In addition, being another enzyme available to hydrolyze acetylcholine, inhibition of BChE by the extracts, especially in the situation of AChE inhibition could further potentiate cholinergic toxicity. Consequently, the inhibitory effect of Jimson weed alkaloid extracts on BChE activity could also contribute in part to their neuromodulatory properties.

The GC-MS characterization of the phytoconstituents in the alkaloid extracts revealed that in addition to the tropane alkaloids (atropine and scopolamine) which have been well characterized in Datura species, other psycho-active compounds such as amphetamine and its derivatives (3-methoxyamphetamine, 3-ethoxyamphetamine, and 3-methyl-amphetamine), cathine, spermine, 3-piperidinemethanol, and phenylephrine among others were also detected. These compounds which are more abundantly distributed in the fruit have been reported to produce varying degrees of neuromodulations. This is in agreement with earlier studies which have reported that alkaloids are more abundantly distributed in the fruit and especially the seed of Jimson weed compared to other parts (Miraldi et al. 2001; Soni et al. 2012). It is also believed that this inequality in distribution is responsible for the higher toxicity and neuromodulation experienced from ingestion of the fruit when compared to other parts of the plant. However, factors such as geographical location, time of harvest, and extraction methods have been reported to influence alkaloid contents of Jimson weed both quantitatively and qualitatively (Mairura and Setshogo 2008; Maheshwari et al. 2013). These factors might also be responsible for the uniqueness of the characterization in this study compared to previous studies. The tropane alkaloids (atropine and scopolamine) are central nervous system modulators, producing effects such as hallucination, depression, and memory impairment (Halpern 2004). Amphetamine and its derivatives have been reported to be MAO inhibitors and can greatly impair monoaminergic neurotransmission to elicit its psychostimulant-like effects (Funada et al. 2014; Santillo 2014). Cathine is identified in our Jimson weed extracts. And its more potent form cathinone has been reported for their neuromodulatory properties and is found present in plants such as khat (Catha edulis) leaves which are traditionally chewed in East African countries for their neurostimulating effects (Gashawa and Getachew 2015). Therefore, it is believed that these phytochemicals could also be responsible for the bioactivities observed in this study.

Conclusion

This study has revealed that the alkaloid extracts from the leaf and fruit of Jimson weed altered the activities of critical enzymes of the monoaminergic and cholinergic neurotransmission systems (in vitro). These modulatory effects could also be part of the mechanisms by which Jimson weed elicit its neuromodulatory effects as reported in folklore. However, the possible contribution of these findings to the physiological manifestations of Jimson weed-induced neuromodulation deserves to be investigated; hence, further in vivo studies are recommended.