Mesembryanthemum tortuosum and Zembrin: Mixed Evidence from In vivo Animal and Clinical Studies on their Antidepressant and Anxiolytic Effects

Abstract

Mesembryanthemum tortuosum is a succulent plant native to southern Africa and was traditionally used to enhance mood. Recent research into M. tortuosum’s mood-enhancing effects has culminated in the development of a standardised extract, Zembrin, containing consistent levels of mesembrine alkaloids considered to be the main bioactive compounds. This review aims to critically investigate published in vivo animal and clinical studies using M. tortuosum, Zembrin, and mesembrine alkaloids, to evaluate the study designs, formulations, and dosages used, and to assess the results in terms of safety and therapeutic efficacy as an antidepressant and anxiolytic. Four databases, namely PubMed, Scopus, Web of Science, and ScienceDirect, were searched using terms relating to ‘clinical trials’, ‘in vivo’, ‘Mesembryanthemum’, ‘M. tortuosum’, ‘pre-clinical’, ‘Sceletium’, ‘S. tortuosum’, and ‘Zembrin’. The search was conducted to identify original research articles published before July 2025. Although published pre-clinical animal and clinical studies on M. tortuosum–derived products offer valuable insights, such as a favourable safety profile and potential efficacy as an anxiolytic and antidepressant, several limitations have been identified, including small sample sizes and studies conducted in clinically irrelevant populations. A limited number of reported outcomes suggest that Zembrin may have positive effects on anxiety, cognitive function, stress resilience, and mood. However, not all studies showed consistent benefits, and some outcomes were limited to specific measures such as EEG changes or reaction time under cognitive stress. Large-scale clinical trials in relevant populations should be conducted to determine the potential of M. tortuosum–derived products as anxiolytics and antidepressants.

Keywords

Aizoaceae - cognitive function - herbal therapeutics - Mesembryanthemum tortuosum - mood enhancement - neuropharmacology - stress resilience

Introduction

Plants have been used medicinally for millennia, with global popularity surging in recent decades regarding the use of herbal medicines. Known for their adaptability to environmental and biological stressors, medicinal herbs exhibit significant phytochemical variability, as demonstrated by studies on plants collected from the wild [1], [2]. To address this variability and the rising demand for herbal medicines, the World Health Organisation (WHO) and other regulatory bodies have issued guidelines emphasising the standardisation of herbal medicines to ensure safety, efficacy, and quality. These guidelines also aim to mitigate risks, such as adulteration, contamination, and inconsistent potency [3], [4]. To adhere to this call for standardisation, several extracts from well-known and popular medicinal herbs have been produced, such as EGb761 from Ginkgo biloba L. (Ginkgoaceae), KSM-66 from Withania somnifera (L.) Dunal (Solanaceae), Ginsana from Panax ginseng C. A.Mey. (Araliaceae), Echinaforce from Echinacea purpurea (L.) Moench. (Asteraceae), and Zembrin from Mesembryanthemum tortuosum (L.) N. E.Br. (syn. Sceletium tortuosum L., Aizoaceae).

Mesembryanthemum tortuosum is a succulent plant native to southern Africa ([Fig. 1 a] and [b]), traditionally used by indigenous peoples to enhance mood and to alleviate abdominal pain and hunger [5]. Over the past two decades, growing interest in M. tortuosum’s antidepressant and anxiolytic effects has culminated in the development of a standardised extract, Zembrin, between 2009 and 2012 [1], [6]. The mood-enhancing effects are primarily attributed to the mesembrine alkaloids (i.e., mesembrine, mesembrenone, mesembrenol, and mesembranol), which are considered to be the plantʼs major active compounds [7]. Mesembrine alkaloids act pharmacologically as antidepressants through selective serotonin reuptake inhibition (SSRI) and phosphodiesterase-4 (PDE4) inhibition [7]. Recent studies also suggest that these alkaloids upregulate vesicular monoamine transporter 2 (VMAT-2) expression and mildly inhibit monoamine oxidase (MAO) [8]. The synergistic actions of the mesembrine alkaloids are therapeutically beneficial, allowing for a significant decrease in required dosage, thereby reducing the possibility of experiencing potential adverse events (AEs) [9].

Fig. 1 a Artistʼs sketch of M. tortuosum (M. de Jong); b inflorescences and leaves of M. tortuosum (Photo: M. de Jong).

While M. tortuosum has been traditionally used by the indigenous peoples of southern Africa since the 1600 s as a mood-enhancing agent [10], it has only recently been scientifically studied using pre-clinical and clinical trials, assisting in regulatory oversight for product development and evaluation [5]. From a dosage perspective, the African Herbal Pharmacopoeia recommends a daily total alkaloid dose of 2 – 12 mg (2000 µg to 12 000 µg) for M. tortuosum [5]. However, clinical studies in healthy populations using Zembrin typically employ far lower doses of total alkaloids, with the highest reported at 200 µg (equivalent to 50 mg Zembrin) [11], [12]. In contrast, a clinical case study successfully used 100 mg Zembrin (400 µg total alkaloids) to treat depression and anxiety without any resulting AEs [5]. Furthermore, in another clinical case study, 400 mg Zembrin was administered twice daily (3200 µg total alkaloids), with no reported AEs, but this highlights the variability in dosing practices [13]. Based on the published literature on pre-clinical in vivo studies of M. tortuosum, it is clear that the optimal effective dose of mesembrine alkaloids has not yet been fully determined. This assertion is supported by a meta-analysis of four clinical trials (117 patients in total) investigating the anxiolytic effect of Zembrin in healthy volunteers, which concluded that there was no statistical difference between the treatment and placebo groups [14].

From a quality-control perspective, most commercially available M. tortuosum products lack standardised extracts, such as Zembrin, leading to inconsistent dosage and efficacy, as well as the potential for experiencing severe AEs for users. Many of these products contain raw M. tortuosum whole plant powder, resulting in significant batch-to-batch dosage inconsistencies. This can further be attributed to the geographical phytochemical variance of the M. tortuosum plant, resulting in variable mesembrine-alkaloid content for each chosen chemovar, as well as the formulation of the product [1]. Mesembrine alkaloids, acting as SSRI and PDE4 inhibitors, may cause herb–drug interactions leading to serious AEs, such as serotonin syndrome, when combined with serotonergic or MAO-inhibiting drugs [6]. This potentially toxic interaction causes serotonin overload and is characterised by confusion, hyperthermia, agitation, hypertension, and tachycardia, underscoring the need for standardised dosing [15]. Consequently, establishing a safe and therapeutically effective standardised dose of M. tortuosum, based on its alkaloid content, is essential.

The pyramid of evidence is a tool that is commonly used to assess the strength and reliability of clinical research based on how likely different study designs are to introduce bias. Some designs offer stronger, more trustworthy evidence and are therefore ranked higher in the pyramid [16]. The pyramid shape also reflects the quantity of available research, with fewer studies typically available at the top [17]. For example, there are typically fewer randomised placebo-controlled clinical studies (RCTs) than pre-clinical studies, such as in vivo animal studies, and even fewer systematic reviews of RCTs. Studies higher up in the pyramid are generally less prone to bias and provide stronger evidence regarding safety and efficacy. For example, clinical studies produce evidence in humans, but their quality still depends on key factors, such as sample size, randomisation, and study duration. In contrast, animal studies allow researchers to perform procedures that are not ethically possible in humans, such as organ dissection to study drug distribution and mechanisms of action. However, results from animal studies offer limited clinical relevance due to biological differences between species and the tendency of the model to oversimplify complex human diseases. Thus, a systematic review, which analyses data from multiple RCTs, is considered the most reliable form of evidence for evaluating the safety and efficacy of a treatment [18].

This narrative review aims to evaluate and discuss published in vivo animal studies and clinical trials using M. tortuosum, Zembrin, or mesembrine alkaloids, while specifically considering their study designs, formulations and mode of administration, and dosages to assess the strength of evidence of their therapeutic efficacy and safety. The key aspects evaluated include the study design and characteristics (e.g., study duration, randomisation, blinding, number of participants, sponsor, etc.), aim of the study (e.g., subjective or objective outcomes), formulation and dosage range of M. tortuosum (namely Zembrin, crude extract, or pure mesembrine alkaloids), route of administration and vehicle used, main outcomes of the study and reported AEs, and strength of evidence regarding safety/efficacy concerning the medical condition investigated. This review differs from previously published comprehensive reviews covering all aspects of M. tortuosum [7] by providing an updated but also focused, in-depth discussion on all in vivo animal and clinical studies conducted using M. tortuosum–derived products. Moreover, it provides recommendations, specifically regarding clinical trials, that should be considered for future studies.

Search Strategy and Selection Criteria

Four databases, namely PubMed, Scopus, Web of Science, and ScienceDirect, were searched using the terms ‘clinical trials’, ‘in vivo’, ‘M. tortuosum’, ‘pre-clinical’, ‘Sceletium’, ‘S. tortuosum’, and ‘Zembrin’. All studies that reported on animal experiments and clinical trials, using any reported M. tortuosum–derived treatment and investigating any biological or clinical outcome were included in this review. The search resulted in the identification of 10 in vivo animal studies, 8 clinical studies, and 1 systematic review of clinical studies published before July 2025. [Fig. 2] provides a PRISMA diagram illustrating the search strategy and results. Three in vivo animal studies published between 2002 – 2006 and written in Japanese could not be retrieved and were therefore excluded. Full-text articles were included if they reported on in vivo animal studies and clinical trials that used raw M. tortuosum plant material, standardised Zembrin, or pure mesembrine alkaloids as the intervention and involved participants, regardless of age, gender, species, or health condition.

Fig. 2 PRISMA flow diagram of the search strategy employed.

Pre-Clinical Animal Studies

A summary of in vivo studies investigating the effects of M. tortuosum–derived interventions is presented chronologically in [Table 1]. A study in male Wistar rats was conducted where a 2 : 1 aqueous ethanolic extract of unfermented M. tortuosum leaves was administered once daily for 17 days by oral gavage [19]. Ninety rats were randomised into three treatment groups receiving either 0.85% saline (placebo) or 5 to 20 mg/kg/day of the M. tortuosum extract (concentration of mesembrine-type alkaloids present in the extract were not supplied). The main result from this study was that the lower dose of 5 mg/kg/day had some positive effects on induced psychological stress, but both treatment groups resulted in an anti-inflammatory reaction or intolerance response. It was established by the authors that M. tortuosum increased Th2 (humoral) anti-inflammatory cytokine IL-10 levels, resulting in a Th1 immune-suppressive effect, thus resulting in an anti-inflammatory reaction (the authors concluding interestingly that it led to an inflammatory reaction). However, it was also mentioned that IL-10 could result in an intolerance response since it has previously been implicated in food hypersensitivity or intolerance. Thus, the author concludes that a more defined dosage range for M. tortuosum needs to be established, as there seems to be a “fine line between a beneficial and detrimental dosage range”.

Table 1 Summary of all in vivo preclinical studies conducted using M. tortuosum, Zembrin, and mesembrine alkaloids in chronological order.

Study ref.	Test subjects	Condition	Formulation	Dosage	Main outcomes	Adverse events
[19]	Male Wistar rats (n = 90)	To evaluate the sedative effects of M. tortuosum, in an in vivo model of psychological stress over 17 days using the elevated plus maze test.	A 2 : 1 extract of unfermented M. tortuosum. Placebo 0.85% sterile saline.	Single dose of M. tortuosum extract (5 or 20 mg/kg) or vehicle-control daily. Oral administration by gavage. (No specifications of total mesembrine-type alkaloids present in the extract used.)	A limited effect of lower dose to decrease restraint-stress-induced self-soothing behaviour and decrease stress-induced corticosterone levels. However, increased IL-1β levels argue against the claim that the plant may act as SSRI.	Both doses used resulted in both an inflammatory or intolerance reaction and varying degrees of Th1 immune suppression.
[20]	Male Sprague–Dawley rats (sample sizes of 4 – 10)	To evaluate behavioural responses in (1) the conditioned place preference (CPP) and (2) hot plate, forced swim, elevated plus, and rotarod tests in male rats after M. tortuosum administration.	Study 1: Amphetamine and haloperidol used as references and M. tortuosum alkaloid extract (1.5% mesembrine), an alkaloid enriched fraction (11.8% mesembrine), and mesembrine (purity not stated) administered intraperitoneally (1 mg/kg). Study 2: Morphine, imipramine, chlordiazepoxide, and muscimol included as references and same M. tortuosum samples as in study 1.	Study 1: Amphetamine (1 mg/kg), haloperidol (0.8 mg/kg), M. tortuosum full alkaloid extract (25, 50, and 100 mg/kg–meaning 375, 750, and 1500 µg/kg mesembrine, respectively), alkaloid enriched fraction (5, 10, and 20 mg/kg–meaning 590, 1180, and 2360 µg/kg mesembrine, respectively), and mesembrine (5, 10, and 20 mg/kg–meaning 5000, 10 000, and 20 000 µg/kg mesembrine, respectively). Study 2: Morphine (5 mg/kg), imipramine (15 mg/kg), chlordiazepoxide (5 mg/kg), and muscimol (2 mg/kg), as well as M. tortuosum full alkaloid extract (100 mg/kg–meaning 1 500 µg/kg mesembrine), alkaloid-enriched fraction (20 mg/kg–meaning 2360 µg/kg mesembrine), and mesembrine (20 mg/kg–meaning 20 000 µg/kg mesembrine).	Mesembrine appears to have analgesic properties without abuse liabilities or ataxia. The M. tortuosum fraction has antidepressant properties but does produce ataxia.	AE reporting was omitted from this study.
[21]	14-day study (n = 50), 90-day study (n = 100) pathogen-free male and female Wistar rats	14-day study: to evaluate the safety of repeated oral administration of Zembrin. 90-day study: to assess the possible health hazards, identification of toxic effects on target organs, of daily oral exposure to Zembrin. Both studies aimed to determine a no-observed-adverse-effect level (NOAEL).	Distilled water was used as the vehicle control for oral administration by gavage of Zembrin (constant dosing volume of 10 ml/kg/bw was used).	14-day study (n = 50): single dose Zembrin in distilled water (25, 75, 250, and 500 mg/mL–meaning 100, 300, 1000, and 2000 µg/mL total alkaloids, respectively) and vehicle-control once daily. 90-day study (n = 100): single dose Zembrin in distilled water (10, 30, 45, and 60 mg/mL–meaning 40, 120, 180, and 240 µg/mL total alkaloids, respectively) and vehicle-control once daily.	14-day study NOAEL: 5000 mg/kg/day (20 000 µg/kg/day total alkaloids). 90-day study NOAEL: 600 mg/kg/day (2400 µg/kg/day total alkaloids). No differences between treated animals and control groups were observed during the functional observational battery in both studies.	No mortality- or treatment-related AEs were observed in male or female Wistar rats in the 14/90-day studies.
[23]	Socially raised male Silver-Laced Wyandotte chicks (sample sizes of 15 – 22)	The effect of alkaloid-enriched fraction of M. tortuosum in the chick anxiety-depression model using social separation and the measurement (recording) of distress vocalisations (DVoc).	Alkaloid-enriched fraction from M. tortuosum containing 11.8% mesembrine. Imipramine dissolved in deionised water served as control with alkaloid fraction dissolved in a solution of 20% Tween 80 and deionised water, which was also used as the vehicle-control.	Experiment 1: (n = 22) vehicle-control, (n = 15) Imipramine (10 mg/kg), and (n = 18) M. tortuosum (10, 20, and 30 mg/kg–meaning 1180, 2360, and 3540 µg/kg mesembrine, respectively). Experiment 2: (n = 21) vehicle-control, (n = 16) Imipramine (10 mg/kg), and (n = 16) M. tortuosum (50, 75, and 100 mg/kg–meaning 5900, 8850, and 11 800 µg/kg mesembrine, respectively).	M. tortuosum at 75 and 100 mg/kg (8850 and 11 800 µg/kg mesembrine, respectively) attenuated DVoc rates in anxiety phase (anxiolytic) but did not affect DVoc rates in depression phase (anti-depressive).	AE reporting was omitted from this study.
[24]	Free-moving adult Fisher 344 rats (n = 18) (crossover design)	To evaluate the cognitive-enhancing effect of Zembrin by analysing spectral power over 8 frequency ranges, namely delta, theta, alpha1, alpha2, beta1a, beta1b, beta2, and gamma power by constructing electropharmacograms.	0.9% NaCl was used as the vehicle for oral administration by gavage of Zembrin (constant dosing volume of 1 ml/kg/bw was used).	Single acute dose of Zembrin 2.5 mg/kg (n = 16), 5.0 mg/kg (n = 16), or 10.0 mg/kg (n = 16)–meaning 10, 20, and 40 µg/kg total alkaloids, respectively, with 0.9% NaCl as vehicle and control (n = 18) (1 ml/kg of vehicle).	Zembrin dose-dependently attenuated all frequency ranges, to varying degrees. The most prominent was the statistically significant reduction in alpha2 and beta1a waves.	AE reporting was omitted from this study.
[25]	Adult male (n = 19) Sprague–Dawley rats	To evaluate the electric excitability of the rat hippocampus (ex vivo and in vitro) in the presence of Zembrin by testing its action in the presence of several receptor agonists or antagonists of glutamatergic transmission.	Artificial cerebrospinal fluid was used as the vehicle-control in both studies. Ex vivo: oral administration by gavage of Zembrin for one week. In vitro: Four major alkaloids tested directly in the hippocampus slice preparation.	Ex vivo: single dose Zembrin (5 mg/kg or 10 mg/kg (20 and 40 µg/kg total alkaloids, respectively) or control daily. In vitro: single acute dose of mesembrine at 3.46/8.65 nmol (1.00/2.50 µg), mesembrenone at 17.14/34.80 nmol (4.45/9.03 µg), mesembrenol at 17.29/34.58 nmol (4.52/9.04 µg), mesembranol at 17.16/34.32 nmol (4.52/9.04 µg), and vehicle-control (ACSF) into hippocampus.	Zembrin ex vivo and all alkaloids in vitro attenuated the amplitude of the population spike during electric stimulation as single shock, as well as theta burst stimulation, with only mesembrenol and mesembranol attenuating AMPA-receptor-mediated transmission.	AE reporting was omitted from this study.
[22]	Adult male Flinders Sensitive Line (FSL) rats (n = 92) and Flinders Resistant Line (FRL) rats as controls (n = 6) to confirm depressive phenotype	To obtain a chromatographic fingerprint of a standardised extract of M. tortuosum (Zembrin) and to evaluate the acute antidepressant-like properties of Zembrin versus the reference antidepressant escitalopram (ESC).	Physiological saline was used as the vehicle control for oral administration by gavage of Zembrin and escitalopram (each API was dissolved in 3 ml of vehicle control).	Single dose of ESC (5, 10, and 20 mg/kg) or Zembrin (5, 10, 25, 50, and 100 mg/kg) (20, 40, 100, 200, and 400 µg/kg total alkaloids, respectively) or vehicle-control was administered 24 h, 6 h, and 1 h before testing in open field test and forced swim test.	FSL rats showed significantly decreased swimming and climbing (coping) behaviours and significantly increased immobility (despair), versus FSL controls, with Zembrin 50 mg/kg (200 µg/kg total alkaloids) being the most effective antidepressant dose, showing equivalence to ESC 5 mg/kg.	AE reporting was omitted from this study.
[26]	Zebrafish larvae (n = 5 per well in a 48-well plate)	To investigate M. tortuosum alkaloids for possible anxiolytic-like effects in the 5-dpf in vivo zebrafish model by assessing thigmotaxis and locomotor activity.	E3 embryo medium 1% DMSO negative control Diazepam positive control Stock solutions for each alkaloid were used.	M. tortuosum alkaloids each at 10, 15, 30, and 50 µM (mesembrine 2.89, 4.34, 8.68, and 14.47 µg/mL, respectively), mesembrenone (2.59, 3.89, 7.78, and 12.97 µg/mL, respectively), mesembrenol (2.61, 3.92, 7.84, and 13.07 µg/mL, respectively), and mesembranol (2.63, 3.95, 7.90, and 13.17 µg/mL, respectively) were prepared in E3 medium daily. Working solution of 2.5, 5, and 10 µM. Diazepam was used as a positive control.	During the dark challenge, the alkaloids exhibited anxiolytic-like effects as evidenced by the reverse-thigmotaxis behaviour of the zebrafish larvae compared to the control group. All alkaloids demonstrated an anxiolytic-like effect; however, mesembrenone and mesembranol demonstrated greater anxiolytic-like activity than mesembrine and mesembrenol.	During the determination of the maximum tolerated concentration, 75 and 150 µM of isolated alkaloids significantly impaired locomotor activity.
[8]	Wild-type zebrafish larvae (n = 120) (dose-response study)	To determine an anxiolytic- and antidepressant-like effective concentration of Zembrin and the isolated major alkaloids using a dose response in zebrafish larvae.	E3 medium was used as the control for this study Zembrin, isolated major mesembrine alkaloids and control were administered via immersion.	Single dose of Zembrin (0.25, 0.5, 1.0, 5.0, 10.0, 12.5, 50.0, 250.0, and 500.0 µg/ml or control (n = 12 per experimental group) was administered, and thereafter an optimal concentration, with one lower and one higher dose, was chosen for subsequent testing, namely Zembrin 5, 12.5, and 25 µg/ml. Total mesembrine-type alkaloid concentrations were calculated using the known percentages in Zembrin and are listed as such.	Zembrin 12.5 µg/ml was the most effective anxiolytic-like concentration as it significantly decreased locomotor activity (without eliciting toxicity), where mesembrine was the only alkaloid producing anxiolytic-like effects at concentrations equal to 12.5 and 25 µg/ml of Zembrin. Zembrin 25 µg/ml reversed the effects of reserpine, indicating antidepressant-like properties.	Zembrin 500 µg/ml elicited toxicity after treatment for 24 h.
[27]	Male (n = 47) and female (n-48) adult Wistar rats	This study investigates the antidepressant and/or anxiolytic effects of a standardised extract of M. tortuosum, Zembrin.	Physiological saline was used as the vehicle control for oral administration by gavage of mesembrine (reference compound), Zembrin, and escitalopram (reference compound).	Single dose of escitalopram (20 mg/kg), mesembrine (7.481 µg/kg, calculated according to its proportional content in Zembrin 12.5 mg/kg), or Zembrin 12.5 and 25.0 mg/kg (50 and 100 µg/kg total alkaloids) daily for 8 weeks.	In males, ZEM12.5 (50 µg/kg total alkaloids) decreased anhedonia- and anxiety-like behaviour, as well as cortical and hippocampal PDE4B, and increased plasma interleukin-10. MES (7.481 µg/kg) induced a transient decrease in anhedonia-like behaviour and increased hippocampal serotonergic and cortical dopaminergic activity, whilst decreasing hippocampal PDE4B, and ZEM25 (100 µg/kg total alkaloids) increased plasma interleukin-10 but decreased cortical glutathione, indicating paradoxical anti-inflammatory and prooxidant effects.	It is reported that in the escitalopram group there was one male mortality for unknown reasons.

The effect of an M. tortuosum extract (1.5% mesembrine) at 25, 50, and 100 mg/kg (375, 750, and 1500 µg/kg mesembrine), an alkaloid-enriched fraction (11.8% mesembrine) at 5, 10, and 20 mg/kg (590, 1180, and 2360 µg/kg mesembrine), and pure mesembrine at 5, 10, and 20 mg/kg (5000, 10 000, and 20 000 µg/kg mesembrine) in male Sprague–Dawley rats was studied [20]. A conditioned place preference (CPP), hot plate, forced swim, elevated plus maze, and rotarod test were conducted. For each test, appropriate reference compounds (amphetamine, haloperidol, morphine, imipramine, chlordiazepoxide, and muscimol, respectively) were used. In the CPP test, only the positive controls showed a significant difference in comparison with the vehicle control (i.e., negative control). In a follow-up study, all samples were tested at the relatively high concentrations of 100, 20, and 20 mg/kg for the extract, enriched fraction, and mesembrine, respectively. In the hotplate test, only mesembrine and morphine showed a statistically significant increase in response latency compared with the vehicle control. In the forced swim test, only imipramine and the enriched fraction showed a significant decrease in float time compared with the vehicle group. In the elevated plus maze test, only chlordiazepoxide showed a significant difference compared with the vehicle control. In the final rotarod test, the positive control and the enriched fraction showed a statistically significant difference in latency to fall compared with the vehicle control. Due to the small sample sizes and the large standard deviation in all test results, it is difficult to come to any definitive conclusion on the effectiveness of the interventions, keeping in mind that only 3 out of the 15 test points (3 interventions in 5 tests) showed a positive result. However, the authors concluded that mesembrine exhibits analgesic properties without abuse liabilities or causing ataxia, whereas the alkaloid fraction did show antidepressant properties but caused ataxia, which supports the conclusion made [19] regarding the seemingly fine line between benefit versus harm (e.g., (anti-)inflammatory or intolerance response). This highlights the possibility that some components within M. tortuosum act as agonists or antagonists against one another and that further research on the isolated compounds may prove beneficial.

The standardised extract, Zembrin, was tested for the first time in Wistar rats [21] and was developed as a standardised M. tortuosum extract. (Gericke et al. [22] conducted a quantitative analysis and reported that the 0.35 – 0.45% of total alkaloids in 25 mg of Zembrin consisted of 47.9% mesembrenone, 32% mesembrenol, 13.3% mesembrine, and 6.8% mesembranol.) Two consecutive studies were conducted in Wistar rats to assess the toxicological effects of Zembrin and to determine a no-observed-adverse-effect level (NOAEL). In the 14-day study, doses ranged from 0 to 5000 mg/kg/day, and results were used to determine the dosage for a follow-up 90-day study, where doses ranged between 0 and 600 mg/kg/day. The NOAEL was determined to be 5000 and 600 mg/kg/day (20 000 and 2400 µg/kg/day total alkaloids) in the 14- and 90-day studies, respectively. This equates to 17 500 – 22 500 and 2100 – 2700 µg/kg/day total alkaloids for the 14- and 90-day studies, respectively. No mortality- or treatment-related AEs were reported in either study. The authors applied a 100-fold uncertainty factor to the NOAEL of 600 mg/kg/day to compensate for species-specific differences and concluded that a daily consumption of up to 420 mg Zembrin can be assumed to be a safe dosage (1680 µg total alkaloids). While this highlights the good safety profile of Zembrin, it poses the question of what the optimal dose of Zembrin would be and whether higher doses should be investigated.

Carpenter et al. [23] investigated the effects of an M. tortuosum alkaloid-enriched fraction (11.8% mesembrine) in a chick anxiety-depression model using male Silver-Laced Wyandotte chicks. The extract, identical to that used by [20], was tested at various dosages. In the first experiment, the alkaloid-enriched fraction was administered at 10, 20, and 30 mg/kg (1180, 2360, and 3540 µg/kg mesembrine), with only imipramine (positive control) showing a significant effect compared with the vehicle control (negative control) during the depression phase. In the second experiment, higher dosages of 50, 75, and 100 mg/kg (5900, 8850, and 11 800 µg/kg mesembrine) were tested, with the enriched fraction demonstrating significant anxiolytic effects at 75 and 100 mg/kg (8850 and 11 800 µg/kg mesembrine) but no effects in the depression phase. Notably, imipramine, administered at the same dose as in the first experiment, failed to show a significant effect in the depression phase, unlike in the first experiment. The authors concluded that the alkaloid-enriched fraction lacked antidepressant effects at the tested doses but exhibited anxiolytic effects at higher doses (75 and 100 mg/kg). The results did not indicate a dose-response curve, with the highest dose slightly less effective than the 75 mg/kg dose. The same trend can be observed in the first study, where no dose-response could be observed. This may be due to the small sample sizes or even mild sedative effects, as shown by [19], [20]. AE reporting was omitted from this study.

Dimpfel et al. [24] evaluated the cognitive-enhancing effects of a single acute dose of Zembrin at 2.5, 5.0, and 10.0 mg/kg (10, 20, and 40 µg/kg total alkaloids) in a crossover study involving 18 freely moving adult Fischer rats. Spectral power was analysed across various frequency ranges to construct electropharmacograms, which were compared with reference data from other botanicals and rolipram, a PDE4 inhibitor, using 0.9% NaCl as the negative control. The study found that Zembrin dose-dependently attenuated all frequency ranges to varying degrees. In a subsequent ex vivo and in vitro study, Dimpfel et al. [25] examined the electrical excitability of hippocampal slices from 19 Sprague–Dawley rats. Zembrin at 5.0 and 10.0 mg/kg (20 and 40 µg/kg total alkaloids) was administered over one week in the presence of glutamatergic receptor agonists such as (S)-(−)-5-fluorowillardine, (±) trans-ACPD, and O-phospho-L-serine, while isolated mesembrine alkaloids were tested at two concentrations in hippocampal slice preparations, using artificial cerebrospinal fluid as the negative control. Both Zembrin and the mesembrine alkaloids reduced the amplitude of the population spike. However, only mesembrenol and mesembranol attenuated AMPA receptor-mediated transmission. These findings underscore Zembrinʼs potential cognitive effects, supported by its standardised alkaloid profile.

Gericke et al. [22] conducted a study on adult male Flinders Sensitive Line (FSL) rats where escitalopram was used as a reference standard (positive control) to evaluate the acute antidepressant-like properties of Zembrin (mesembrine at 13.2% of total alkaloids). Zembrin at 5, 10, 25, 50, and 100 mg/kg (20, 40, 100, 200, and 400 µg/kg total alkaloids), escitalopram at 5, 10, and 20 mg/kg or physiological saline (negative control) were administered 24, 6, and 1 h before open field and forced swim testing were conducted. It was concluded that Zembrin at 25 and 50 mg/kg (100 and 200 µg/kg total alkaloids) and escitalopram at 5 mg/kg were effective antidepressants based on the acute dosing regimen used in this study. Moreover, it was established that Zembrin at a dose of 50 mg/kg (200 µg/kg total alkaloids) therapeutically equates to a 5 mg/kg dosage of escitalopram. However, at the highest dose tested (100 mg/kg) (400 µg/kg total alkaloids), a marked decrease in specific test parameters (e.g., time spent immobile, swimming, and struggling in the forced swim test and locomotor activity in the open field test) was observed as compared with the ‘optimal’ dose of 50 mg/kg, which may indicate that the effect does not appear to be dose-dependent or that toxicity may appear at this dosing level. This contradicts the good safety profile that was reported in Wistar rats as published by Murbach et al. [21].

Maphanga et al. [26] evaluated the anxiolytic-like effects of the major alkaloids found in M. tortuosum by assessing thigmotaxis and locomotor activity in a zebrafish model. Stock solutions of 10, 15, 30, and 50 µM were prepared in E3 medium for each mesembrine alkaloid (mesembrine at 2.89, 4.34, 8.68, and 14.47 µg/mL, mesembrenone at 2.59, 3.89, 7.78, and 12.97 µg/mL, mesembrenol at 2.61, 3.92, 7.84, and 13.07 µg/mL, and mesembranol at 2.63, 3.95, 7.90, and 13.17 µg/mL, respectively). DMSO (1% v/v) was used as a negative control and diazepam (2.5, 5, and 10 µM) as the positive control. It was concluded that all the alkaloids elicit anxiolytic-like activity by exhibiting reverse-thigmotaxis behaviour as compared with the control. It was also stated that mesembrenone and mesembranol exhibited greater anxiolytic-like activity compared with the other mesembrine alkaloids.

Another in vivo study was conducted by Gericke et al. [8], who used a wild-type zebrafish larvae model in a dose-response study using Zembrin, isolated mesembrine-alkaloids, and E3 control medium to determine the effective concentration for anxiolytic and antidepressant-like effects (mesembrine-type alkaloid concentrations were calculated using the known percentages in Zembrin and are listed as such [22]). A single dose of Zembrin was administered in concentrations ranging from 0.25 µg/ml to 500.0 µg/ml, where 500.0 µg/ml resulted in toxicity after 24 h of treatment. Concentrations of 5.0, 12.5, and 25.0 µg/ml were chosen for subsequent testing. The authors concluded that Zembrin 12.5 µg/ml resulted in optimal anxiolytic-like effects, while Zembrin 25.0 µg/ml indicated antidepressant-like qualities by reversing the effects of reserpine. Mesembrine was the only major alkaloid to produce anxiolytic-like effects at concentrations equal to those of Zembrin at 12.5 and 25.0 µg/ml.

The most recent in vivo study was published by Gericke et al. [27]. Ninety-five adult male and female Wistar rats were used to create an unpredictable chronic mild stress (UCMS) model, to test the antidepressant and/or anxiolytic abilities of the standardised extract Zembrin (12.5 or 25.0 mg/kg/day) (50 and 100 µg/kg/day total alkaloids) versus reference compounds, namely, escitalopram (20 mg/kg/day) or mesembrine, calculated according to its proportional content in Zembrin at 12.5 mg/kg/day (7.5 µg/kg/day mesembrine), due to the assertion that it might be a significant contributor to M. tortuosum’s anxiolytic effects. Behavioural analyses were conducted by assessing anxiety in the elevated plus maze test, and movement was analysed in the open field test (OFT). Cognition and anxiety were assessed in the Barnes maze test, behavioural despair was evaluated in the forced swim test (FST), and anhedonia was analysed in the sucrose preference test (SPT). Interestingly, treatment effects were only observed in the male rats, and it was concluded that Zembrin at 12.5 mg/kg/day (50 µg/kg/day total alkaloids) reduced signs of anhedonia and anxiety, lowered PDE4B levels in both the cortex and hippocampus, and increased levels of the anti-inflammatory marker IL-10 in the blood. Mesembrine created a short-term improvement in anhedonia-like behaviour and boosted serotonergic activity in the hippocampus and dopaminergic activity in the cortex, while also reducing PDE4B in the hippocampus. Zembrin at 25 mg/kg/day (100 µg/kg/day total alkaloids) raised IL-10 levels but lowered glutathione in the cortex, suggesting both anti-inflammatory and pro-oxidant effects.

Clinical Studies

A summary of clinical studies evaluating the effects of M. tortuosum or Zembrin is presented chronologically in [Table 2]. In a clinical study, 16 healthy male and female participants were given a single identical dose of 25 mg Zembrin (100 µg total alkaloids) or placebo (inert excipients), with a crossover occurring after 5 – 9 days, to evaluate its anxiolytic potential using a double-blind, placebo-controlled, crossover design [28]. A perceptual-load task and an emotion-matching task were completed by all participants while undergoing magnetic resonance imaging (MRI) (participants were scanned 2 h post administration), to evaluate the activity in the amygdala and hypothalamus. It was concluded that Zembrin at 25 mg (100 µg total alkaloids) decreased anxiety-related amygdala reactivity and reduced amygdala-hypothalamus coupling. However, from Figure 2a and 3a in Terburg et al. [28], no statistical difference between the treatment and control groups was evident. Moreover, both groups demonstrated large standard errors, making interpretation of the perceptual-load task test difficult and emphasising the inflation of mechanistic outcomes without providing evidence of a resulting clinical effect. Participants were required to report any experienced AEs, but only very minor and somewhat subjective AEs were reported, such as feeling ‘a bit woozy’.

Table 2 Summary of all clinical studies conducted using M. tortuosum, Zembrin, and mesembrine alkaloids in chronological order.

Reference and study design	Participants	Condition	Formulation	Dosage	Main outcomes	Adverse events
[28] Double-blind, placebo-controlled, crossover design	16 healthy female (n = 8) and male (n = 8) participants. Mean age = 19.2 years.	Anxiolytic potential of Zembrin 5 – 9 days apart.	Both Zembrin and placebo were in identical gelatin capsules.	Single acute dose of Zembrin 25 mg (100 µg total alkaloids) or placebo with crossover after 5 – 9 days.	Zembrin reduces anxiety-related amygdala reactivity and attenuates amygdala–hypothalamus coupling as compared to placebo effects.	Participants were asked to report AEs, but minor subjective experiences were recorded.
[29] Randomised, double-blind, parallel-group, placebo-controlled single-centre study	37 healthy male and female participants between 18 and 55 years of age (3 withdrew).	Safety and tolerability of Zembrin at two different dosages over three months.	Both Zembrin and placebo were in identical capsules.	Single dose of Zembrin 8 mg (32 µg total alkaloids) daily (n = 12), 25 mg (100 µg total alkaloids) daily (n = 12), and placebo daily (n = 13).	Safety and tolerability across all dosages of Zembrin with no apparent differences between the three treatments regarding vital signs, 12-lead ECG, haematological, or biochemistry parameters.	Participants were asked to report AEs in a diary: subjects reported headaches, abdominal discomfort, loss of appetite, and muscle spasms. The placebo group reported more AEs than the treatment groups.
[30] Randomised, placebo-controlled, 3-week crossover design (9 weeks total)	21 healthy female (n = 12) and male (n = 9) participants. Mean age = 54.6 years (five subjects dropped out).	Evaluate the neurocognitive effects and assess the safety and tolerability of Zembrin and its implications for Alzheimerʼs and dementia over 9 weeks.	Both Zembrin and placebo were in identical capsules.	Single dose of Zembrin 25 mg capsule (100 µg total alkaloids) daily for 3 weeks, washout for 3 weeks, then a single dose of placebo daily for the last 3 weeks.	Zembrin significantly improved cognitive flexibility and executive function. No significant differences in the other seven measurements were recorded, with the neurocognitive index non-significantly favouring the placebo.	Zembrin group reported transient gastrointestinal discomfort whereas the placebo group complained of more frequent AEs.
[11] Double-blind, randomised, placebo-controlled, 3-armed parallel design	60 healthy female (n = 30) and male (n = 30) participants. Mean male age = 51.53; Mean female age = 51.93.	Examine differences in 102 EEG parameters and differences in 9 psychometric tests after a single acute dose of Zembrin.	Both Zembrin and placebo were in identical capsules.	Single acute dose of Zembrin 25 mg (100 µg total alkaloids) or 50 mg (200 µg total alkaloids) or placebo.	Zembrin induced enhanced increases in power of frontal delta, theta, alpha1, and alpha2 frequencies during several tasks and significant differences in EEG parameters and non-significant differences in all psychometric tests.	Participants were asked to report AEs, but none were reported.
[12] Randomised, double-blind, placebo-controlled, 3-armed parallel design	60 healthy female (n = 28) and male (n = 32) participants. Mean male age = 59.7 years; mean female age = 56.7 years.	Effect of Zembrin on brain electrical activity over six weeks.	Zembrin and a placebo control.	Single dose of Zembrin 25 mg (100 µg total alkaloids) or 50 mg (200 µg total alkaloids) or placebo daily.	Zembrin showed significant activity on three levels of evidence: questionnaires, psychometry, and quantitative EEG. The results indicate that in healthy people Zembrin improves some aspects of cognitive function, decreases anxiety, and may enhance mood.	No AEs were reported in this manuscript.
[31] Randomised, placebo-controlled, parallel design	60 healthy and active female (n = 12) and male (n = 48) participants 20 to 35 years of age.	The effect of Zembrin on reactive agility, visual tracking, and mood over 8 days.	Both Zembrin and placebo were in similar capsules.	Single dose of Zembrin 25 mg (100 µg total alkaloids) or placebo daily.	Zembrin significantly improved reactive performance to a visual stimulus when a cognitive stress was added to the task but failed in all other test measures.	Participants were asked to report AEs, but none were reported.
[32] Placebo-controlled, double-blind, between-subject experimental design	Study 1: 20 healthy female (n = 14) and male (n = 6) participants. Mean age = 19.6 years. Study 2: 20 healthy female (n = 9) and male (n = 11) participants Mean age = 21.3 years.	Study 1 measured subjective experiences of mood, and study 2 measured subjective experiences of anxiety and physiological indicators of stress; both studies measured at baseline, pre-stress induction, and post-stress induction.	Zembrin and a placebo control.	Single acute dose of Zembrin 25 mg (100 µg total alkaloids) or placebo.	Study 1: no effect. Study 2: ameliorate subjective and physiological indicators of stress/anxiety in young healthy volunteers.	Participants were asked to complete subjective self-report measures, but none reported their anxiety high on the given scale.
[33] Placebo-controlled, double-blind, between-subject experimental design	16 non-resistance-trained females (n = 16). Mean age = 21.0 years.	The effect of Zembrin supplementation for three days on muscle soreness, markers of muscle damage, mood, and exercise performance.	Both Zembrin and placebo were in similar capsules.	Single dose of Zembrin 25 mg (100 µg total alkaloids) (n = 8) or placebo (n = 8) daily.	Zembrin decreased perceived soreness (possibly due to reduced anxiety) and preserved range of motion and more favourable overall mood.	Participants were asked to report AEs, but none were reported.

In another clinical study, 37 healthy male and female participants were given a single dose of Zembrin at 8 mg (32 µg total alkaloids), 25 mg (100 µg total alkaloids), or placebo (lactose monohydrate, sodium starch glycolate, and magnesium stearate) daily to evaluate safety and tolerability over three months in a randomised, double-blind, parallel-group, placebo-controlled single-centre study [29]. It was concluded that Zembrin exhibited a good safety and tolerability profile at both dosages with no significant changes in participant vital signs, electrocardiograms (ECG), body weight, haematological, or biochemical parameters compared with the placebo. However, three participants withdrew from the study due to experiencing headaches and abdominal pain. Other AEs were reported by the participants in their diaries such as constipation, loss of appetite, insomnia and muscle spasms but more frequently in the placebo group than in the treatment groups, yet it was concluded by the authors that these AEs could be related to the investigational medicinal product (IMP), which contradicts the fact that more AEs were reported in the placebo group than in the Zembrin group.

A total of 21 healthy female and male participants were subjected to a randomised placebo-controlled 3-week crossover study to evaluate the neurocognitive effects of 25 mg Zembrin (100 µg total alkaloids) and the implications for Alzheimerʼs and dementia [30]. Participants were administered a single dose of Zembrin or placebo once daily for three weeks, followed by a 3-week washout before the crossover for the second 3-week treatment period, and were monitored during the entire 9-week period. During this study, two participants dropped out during the placebo phase and three participants dropped out during the Zembrin phase. Participants taking Zembrin reported AEs such as transient gastrointestinal discomfort, but it was stated that the placebo group reported more frequent AEs. It was concluded by the authors that Zembrin significantly improved cognitive flexibility and executive function, but no significant difference was found in the seven other test indices. The overall neurocognitive index (average of all nine test parameters) does, however, show a slight but non-significant advantage for the placebo group. Moreover, baseline values were only reported at time zero, but none were reported in week 6 before initiating the crossover phase, which makes interpretation of the overall results challenging.

Dimpfel et al. [11] conducted a double-blind, randomised, mannitol-placebo-controlled, three-armed study with parallel design on 60 healthy female and male participants, assessing the effects of a single acute dose of Zembrin at 25 (100 µg total alkaloids) and 50 mg (200 µg total alkaloids) by examining the differences in 102 EEG parameters and 9 psychometric tests 120 min after intake of placebo or Zembrin. It was concluded that Zembrin increased the power of frontal delta, theta, alpha1, and alpha2 frequencies during several tasks and resulted in significant differences in EEG parameters but no significant differences in psychometric test results, highlighting the overinterpretation of mechanistic outcomes in the absence of a significant clinical effect. Participants were required to report AEs, but none were reported. In a follow-up study, the same study design was used to evaluate the effects of Zembrin on brain electrical activity over a six-week period [12]. Zembrin was administered at a dosage of 25 mg or 50 mg daily (100 or 200 µg total alkaloids). Participants were asked to complete the Profile of Mood States and Hamilton Anxiety Rating Scale questionnaires while undergoing a quantitative EEG. All tests were performed 1 h after administration. The authors concluded that Zembrin improves certain aspects of cognitive function, decreases anxiety, and possibly enhances mood. No AEs were reported in this manuscript.

In a clinical study, 60 healthy active female participants were given either a placebo (corn maltodextrin and rice protein) or 25 mg Zembrin (100 µg total alkaloids) over eight days to evaluate the effects of Zembrin on mood, reactive agility, and visual tracking [31]. Participants were required to report any experienced AE, but none were reported. Screening of participants was conducted one day before and after the administration period. It was concluded by the authors that Zembrin significantly improved reactive performance to visual stimuli in the presence of cognitive stress, but non-significant differences in all other test measures were recorded.

Reay et al. [32] utilised a placebo-controlled, double-blind, between-subject experimental design to conduct two studies, each including 20 healthy male and female participants. Study 1 measured the subjective experiences of mood after administration of 25 mg Zembrin (100 µg total alkaloids) or placebo, and it was concluded that there was no statistical difference. Study 2 measured the subjective experiences of anxiety and physiological indicators of stress after administration of 25 mg Zembrin or placebo. Participants were screened at baseline, pre-stress, during stress, and post-stress for both studies. The authors concluded that Zembrin ameliorates subjective and physiological indicators of anxiety and stress. Participants were asked to complete subjective self-reporting measures, with none reporting a ‘high’ on the given scale. The authors concluded that the provided ‘stressor’ might have been too mild and therefore recommended changes in study designs for future studies.

A double-blind, between-groups study design was used to evaluate the effects of Zembrin on muscle soreness, muscle damage, exercise performance, and mood [33]. Sixteen non-resistance-trained females participated in this study and were either given a placebo (gluten-free cornstarch) or 25 mg Zembrin (100 µg total alkaloids) daily for three days following exercise at the baseline visit, one day post-exercise, and 30 min before returning for the 48 h follow-up visit. Participants were required to report any experienced AEs, but none were reported. The authors concluded that Zembrin decreased perceived muscle soreness, preserved range of motion, and improved the overall mood.

Critical Evaluation

In vivo animal studies

The current evidence from pre-clinical in vivo animal studies investigating the safety and efficacy of M. tortuosum–derived treatments presented both encouraging and cautionary findings. When evaluating these studies, it is evident that there are inconsistencies in study design, extract formulation, dosing, and inadequate blinding, which limit the strength of evidence regarding both therapeutic efficacy and safety.

Although [19] successfully employed randomisation and included 90 rats across treatment groups, no positive control was included, and no comprehensive chemical characterisation of the M. tortuosum extract was performed, with only mesembrine qualitatively identified as a minor component. It was concluded that after the administration of M. tortuosum, IL-1β increased. IL-1β is known to increase serotonin uptake; thus, it would be expected that the levels of this cytokine would decrease after M. tortuosum treatment since it is considered to act as an SSRI, but this was not observed. The increase in the anti-inflammatory cytokine IL-10, however, supports the theory that M. tortuosum acts as an SSRI, since it has been shown that antidepressants such as fluoxetine, which have a serotonergic mode of action, increase the production of IL-10 [34]. Furthermore, the author acknowledged the need for defining a safe and effective dosage range, noting a potential fine line between therapeutic benefit versus harm.

Loria et al. [20] used robust positive controls such as amphetamine, haloperidol, morphine, imipramine, and chlordiazepoxide, strengthening the validity of their findings. While pure mesembrine demonstrated notable analgesic effects without causing ataxia, the enriched fraction displayed antidepressant-like effects but induced ataxia, highlighting the potential antagonistic interactions among the mesembrine alkaloids. Nonetheless, small sample sizes undermine statistical relevance, and inadequate blinding raises further concerns regarding bias. The absence of a clear dose-response relationship across tests aligns with earlier concerns raised by [19] regarding the narrow therapeutic window and possible adverse effects at higher doses. It is also important to note that only 3 of the 15 tests provided a statistically significant difference in favour of the intervention, and, combined with small sample sizes and absence of a clear dose-response relationship, these findings might be due to chance.

Both a 14-day and 90-day study were performed by Murbach et al. [21], whereas most of the in vivo studies lasted less than 24 h, limiting insights into long-term toxicity, drug tolerance, and dependence. Murbach et al. [21] demonstrated an impressive NOAEL of 5000 mg/kg/day (14 days) and 600 mg/kg/day (90 days) (20 000 and 2400 µg/kg/day total alkaloids), corresponding to substantial alkaloid intake, with the equivalent dosage in a 70 kg human being 420 mg Zembrin (1680 µg total alkaloids). In a previously mentioned case study, a psychopharmacologist prescribed a dosage of 400 mg Zembrin twice daily to a patient (3200 µg total alkaloids), and no side effects were reported [13]. These dosages exceed those used in clinical studies, where a maximum of 50 mg Zembrin (200 µg total alkaloids) was used. The African Herbal Pharmacopoeia states that a dosage of 2 – 12 mg mesembrine alkaloids (2000 to 12 000 µg) should be taken daily [5]. This research established that Zembrin and/or M. tortuosum has a good safety profile; however, the large variability in dosage recommendations underlines the fact that an optimal effective dosage should still be defined. It is of interest to note that the study of [19] is cited by Murbach et al. [21] to conclude that “…and an in vivo study of an uncharacterized extract of S. tortuosum in rats demonstrated a positive outcome on the behavioural effects of restraint stress”, even though it was concluded by [19] that both doses (5 and 20 mg/kg/day of alkaloid extract) appeared to increase levels of the anti-inflammatory cytokine IL-10.

Carpenter et al. [23] conducted a study where an alkaloid-enriched fraction identical to that of [20] was administered at different doses. Notably, the expected dose-response relationship was absent, and imipramine (positive control) yielded inconsistent results across experiments. This highlights the importance of using positive controls, with, in this case, the positive control invalidating the results. The absence of adverse event reporting further weakens confidence in these findings, underscoring the limitations of small sample sizes and potential sedative or toxic effects at higher doses, as mentioned by Smith [19].

Dimpfel et al. [24], [25] utilised a more mechanistic approach, obtaining results that provide valuable insight into the neurophysiological mechanisms potentially enhancing cognitive effects; however, small sample sizes and inadequate blinding limit the strength of evidence.

Gericke et al. [22] concluded that at the highest dose of Zembrin, 100 mg/kg (400 µg/kg total alkaloids), a notable reduction in locomotor activity and behavioural responses suggested potential toxicity or lack of efficacy, reinforcing concerns regarding dose-dependent adverse effects and the narrow therapeutic window, as established earlier by [19], [20]. The results at 50 mg/kg (200 µg/kg total alkaloids) did, however, show promising results and were therapeutically equated to a 5 mg/kg dosage of escitalopram.

Expanding beyond mammalian models, [8], [26] assessed the anxiolytic properties of isolated mesembrine alkaloids in zebrafish. It was noted by Gericke et al. [8] that mesembrine alone produced anxiolytic effects comparable to the whole extract, further highlighting the importance of research into individual alkaloids and their therapeutic effects. As mentioned above, [20] concluded that mesembrine exhibits analgesic properties without abuse liabilities or causing ataxia, whereas the alkaloid fraction showed antidepressant properties but caused ataxia. This further suggests that the pure isolated mesembrine alkaloids should be further investigated.

The most recent in vivo study by Gericke et al. [27] confirms that Zembrin increases levels of the anti-inflammatory cytokine IL-10, supporting its proposed immunomodulatory effects as stated by Smith [19]. Although it was confirmed that Zembrin increased plasma levels of IL-10, Zembrin at 25 mg/kg/day (100 µg/kg/day total alkaloids) paradoxically increased cortical oxidative stress markers, indicating potential pro-oxidant effects at higher doses. A notable finding from this study was the significant difference between male and female Wistar rats across most bio-behavioural and biochemical parameters, indicating that male Wistar rats are more sensitive to the UCMS model. Results indicated no statistical differences between the treatment and controls in the female rats, subsequently reducing sample size and limiting statistical power and generalisability of the findings. Contrary to expectation, UCMS increased swimming and reduced immobility, raising further challenges to its utility for modelling immobility-based depression. The 8-week study used escitalopram as a positive control, allowing for thorough comparison in terms of safety and efficacy. Interestingly, escitalopram failed to reverse any of the UCMS-induced behavioural or biochemical changes in male rats. This questions its utility as a positive control, particularly considering that the authors acknowledge that anhedonia-like symptoms are extremely difficult to treat with first-line antidepressants. Additionally, only one mortality was reported during the study in a male rat from the escitalopram group (cause unknown), offering preliminary reassurance regarding the safety profile of Zembrin under the tested conditions.

Collectively, these in vivo studies indicate that both M. tortuosum, Zembrin, and mesembrine alkaloids potentially exhibit anxiolytic, antidepressant, analgesic, and cognitive-modulating properties. However, inconsistent study designs, variable extract formulations, a lack of comprehensive chemical profiling in earlier studies, a lack of blinding, and frequent omission of adverse event reporting limit confidence in the reproducibility of these findings. The notable lack of a consistent dose-response curve across studies further highlights the importance of rigorous study design and formulation. The so-called “irreproducibility crisis”, where it was shown that 75 – 90% of preclinical studies cannot be reproduced, could be considered as applicable here [35].

Many studies, using sample sizes smaller than 30, focused exclusively on male animals, excluding one study where the sex of the adult Fischer rats used was not stated. This approach reduces statistical power and may lead to false-positive or -negative results, particularly given known sex differences in drug metabolism and neurochemistry as indicated by the study of Gericke et al. [27]. Most of these in vivo studies were conducted on rats, with Carpenter et al. [23] using Silver-Laced Wyandotte chicks and Maphanga et al. [26] and Gericke et al. [8] using zebrafish larvae. While rats are commonly used in pre-clinical research, they do not fully model human psychiatric conditions and are often administered doses that exceed those used in humans [36]. This can introduce bias in behavioural outcomes and limit the reliability of conclusions regarding clinical efficacy in humans. Moreover, the use of diverse species, such as rats, chicks, and zebrafish, provides a broader understanding of the pharmacological profile of M. tortuosum, offering comparative evidence across models, although they do not fully model human psychiatric conditions.

Several in vivo studies employed dose-ranging designs, providing critical insight into both the therapeutic window and potential toxicity thresholds. Furthermore, the inclusion of positive controls improves the strength of evidence, allowing for comparative assessment of efficacy relative to known pharmacological agents.

The preclinical safety studies, particularly the work by Murbach et al. [21], established a high NOAEL for Zembrin, laying the foundation for potential clinical dosing guidelines and highlighting the good safety margin in animal models. However, it provides little insight into optimal therapeutic doses or efficacy. Additionally, four studies administered an M. tortuosum extract, with limited quality control regarding its mesembrine-alkaloid content, which, with the well-known variability in plant chemotype, extraction methods, and formulations, may introduce additional bias and/or false positives/negatives. Moreover, inconsistent mesembrine-alkaloid dosing complicates cross-study comparisons, interpretation, and reproducibility of findings.

The literature from in vivo animal studies tends to have a greater level of bias than clinical studies. This can be attributed to the fact that human disease and physiology are not effectively and accurately represented by animal models. It has been established that humans have higher blood–brain-barrier (BBB) permeability than rodents, leading to inadequate drug delivery during animal studies and resulting in a poor translation rate to human studies [37]. Additionally, animal studies usually lack adequate blinding and randomisation [38]. Although many in vivo animal studies are conducted primarily to obtain pharmacological knowledge for academic use and not with the end goal of translating to human models, more than 115 million animals are sacrificed annually in biomedical experimentation. The failure rate of translating drugs that were tested pre-clinically in animal studies to human clinical studies is currently 90 – 92% [39], [40], [41].

Historically, the assessment of safety and efficacy of pharmaceuticals relied greatly on the use of animal models, especially considering that certain experimental models cannot ethically be conducted in humans; this is no longer necessary, as numerous new non-animal methodological approaches have been identified and have been proven to be more clinically predictive than animal testing [42]. The value of pre-clinical animal studies for Zembrin and M. tortuosum should therefore be cautiously interpreted when considering translation to human models, and the overall evidence that they provide regarding safety and efficacy should be considered limited, although the results from these studies have provided evidence of potential pharmacological activity. Properly designed clinical studies in human populations are of far greater importance and will be discussed in the next section.

Clinical studies

The clinical investigation of M. tortuosum, primarily using the standardised extract Zembrin, has yielded preliminary evidence for its potential anxiolytic, cognitive-enhancing, and mood-modulating properties. However, evaluation of the existing clinical studies reveals significant methodological limitations, outcome inconsistencies, and concerns regarding the strength of evidence, which must be considered when interpreting the findings.

The earliest clinical evaluation by Terburg et al. [28] utilised a double-blind, placebo-controlled, crossover design to explore the anxiolytic effects of Zembrin. Although the authors reported reduced amygdala reactivity and decreased amygdala–hypothalamus coupling as indicators of anxiolytic action, inspection of the data (Figures 2a and 3a in Terburg et al. [28]) suggests no clear statistical differences between treatment and placebo groups, while [Table 1] contradicts the visual representation provided in the figures. Moreover, large standard errors in both groups weaken the interpretability of these findings.

Nell et al. [29] conducted a more extensive randomised, double-blind, parallel-group, placebo-controlled trial evaluating the safety of Zembrin. While the study concluded that Zembrin exhibited a favourable safety profile, inconsistencies were noted in the adverse event reporting. Most AEs were paradoxically more frequently reported in the placebo group, with two participants (placebo group) withdrawing due to headaches and abdominal pain. It was concluded by the investigators that most AEs were unrelated to study medication and occurred more frequently in the placebo treatment group.

The study conducted by Chiu et al. [30] investigated the neurocognitive effects of Zembrin by using a placebo-controlled, crossover design over nine weeks. Despite the use of an appropriate design, the study suffered from several methodological weaknesses, including incomplete reporting of baseline values before the crossover phase and a relatively high dropout rate. While improvements in cognitive flexibility and executive function were observed, these were limited to isolated instances, with no significant effects across seven other cognitive parameters. Interestingly, the overall neurocognitive index slightly favoured the placebo group, albeit non-significantly, further highlighting the limitations of the studyʼs validity. Adverse events were reported but were transient and, contrary to expectations, occurred more frequently in the placebo group.

Dimpfel et al. [11] assessed the acute effects of Zembrin on brain activity and cognitive performance using a double-blind, placebo-controlled, parallel-group design. While significant EEG changes, including increased frontal delta, theta, and alpha frequencies, were observed, the neurophysiological alterations did not translate into statistically significant improvements in psychometric tests. The same research group conducted a follow-up study using a similar design over six weeks to evaluate the cognitive and mood-related effects of Zembrin over a longer period [12]. Despite positive conclusions regarding mood enhancement and reduction in anxiety, results were based on subjective self-reporting measures (Profile of Mood States Questionnaire and the Hamilton Anxiety Rating Scale) and EEG alterations, with no corresponding behavioural or subjective measures of anxiety to support an anxiolytic effect. This emphasises the overinterpretation of mechanistic outcomes by the authors without evidence of clinical effects.

Hoffman et al. [31] expanded the scope of Zembrin research to include athletic performance outcomes in a double-blind, placebo-controlled study. While Zembrin significantly improved reactive agility under cognitive stress, no significant effects were observed for mood or visual tracking. Although no AEs were reported, the short duration, small sample size, and lack of dose variation limit broader interpretation regarding efficacy or safety beyond the observed outcomes. It is of interest that Hoffman et al. [31] state that a study conducted by Chiu et al. [30] resulted in “…improvements in mood and in cognitive function, including improvements in composite, verbal, and visual memory, reaction time, processing speed, executive function, and complex attention”, whereas Chiu et al. [30] only concluded improvement in cognitive flexibility and executive function. It is evident that not all studies showed consistent benefits, and some outcomes were limited to specific measures such as EEG changes or reaction time under cognitive stress, with no significant evidence of clinically effective outcomes.

Reay et al. [32] performed two separate double-blind, placebo-controlled studies focusing on subjective mood, anxiety, and physiological stress responses. The first study reported no significant differences in mood outcomes between Zembrin and placebo groups. The second study suggested that Zembrin ameliorated subjective and physiological indicators of anxiety and stress. However, the authors acknowledged that the stressor applied was too mild to produce reliable variation in results, calling the strength of evidence of these findings into question.

The most recent clinical study by Berry et al. [33] evaluated Zembrinʼs effects on post-exercise muscle soreness, range of motion, and mood using a double-blind, placebo-controlled, between-groups design. While Zembrin reduced perceived muscle soreness and preserved range of motion, mood improvements were subjectively self-reported, and the small sample size greatly limits generalisability.

The existing clinical studies investigating the effect of Zembrin show several methodological strengths that improve the understanding of its safety and potential efficacy in humans. A noteworthy aspect is the consistent use of randomised, double-blind, placebo-controlled designs, as demonstrated across several studies [28], [29], [30]. This rigorous approach strengthens the reliability and internal validity of the findings. Additionally, some studies implemented dose escalation strategies, contributing to the understanding of the dose-response relationship and tolerability at varying levels. Notably, most clinical studies consistently reported minimal to no adverse events, reinforcing the favourable safety profile of Zembrin, although many studies (6 out of 8) relied on self-reporting measures of psychological outcomes and AEs, resulting in subjective and variable results.

The observed consistency in certain mechanistic outcomes, such as reduced amygdala reactivity [28] and modulation of brain electrical activity [11], [12], provides potential converging evidence for Zembrinʼs neuroactive properties, supporting its potential as a novel botanical for stress modulation and cognitive enhancement, although all studies were conducted in healthy male and female adult participants, whereas treatment outcomes may be different in clinically diagnosed populations. One study was conducted in only adult female participants, reducing the statistical power of the results, owing to known sex differences in behaviour, drug metabolism, and neurochemistry [33]. The predominant use of healthy participants limits translation to clinical populations with anxiety, depression, or cognitive impairment, which remain the primary target indications for Zembrin.

Additionally, several studies reporting positive findings were sponsored or conducted by companies that have a commercial interest in M. tortuosum products. This in itself does not necessarily imply that the studies are less reliable, but it must be kept in mind that the ‘sponsorship effect’ has been shown to exist in clinical studies [43]. Evidence is less biased if independent researchers who have no financial interest in the outcome can replicate the same results in similar conditions and omit any selective reporting of positive results.

To critically appraise the strength of the existing evidence, a risk-of-bias assessment was performed on all included articles. The in vivo animal studies were assessed using the SYRCLE risk-of-bias tool as seen in [Table 3], and the clinical studies were assessed using the Cochrane risk-of-bias tool (RoB-2) as seen in [Table 4] [44], [45]. A high risk of bias was given to those studies where a clear conflict of interest between the researchers and the manufacturers of Zembrin was present. Some concern of bias was given to studies where a conflict-of-interest statement was missing, as well as in other domains where it was not outright stated how blinding and randomisation was conducted or where selective reporting was observed.

Table 3 Risk-of-bias assessment using the SYRCLE tool for in vivo animal studies.

Study	Randomisation and allocation process	Baseline characteristics	Allocation concealment	Blinding of subjects and personnel	Incomplete outcome data	Selective reporting	Conflicts of interest
[19] Smith, 2011	Low	Low	Some concern	High	Low	Low	Some concern
[20] Loria et al. 2014	Some concern	Some concern	Some concern	High	Low	Some concern	Low
[21] Murbach et al. 2014	Low	Low	Some concern	Some concern	Low	Low	High
[23] Carpenter et al. 2016	Low	Low	Low	Some concern	Low	Some concern	Low
[24] Dimpfell et al. 2016 (b)	Low	Low	Low	Some concern	Low	Some concern	High
[25] Dimpfell et al. 2018	Low	Low	Low	Some concern	Low	Some concern	High
[22] Gericke et al. 2022	Low	Low	Low	Low	Low	Some concern	High
[26] Maphanga et al. 2022	Low	Low	Low	Low	Low	Low	Some concern
[8] Gericke et al. 2024	Low	Low	Low	Low	Low	Low	Some concern
[27] Gericke et al. 2025	Low	Low	Low	Low	Low	Low	Some concern

Table 4 Risk-of-bias assessment of clinical studies using the Cochrane tool.

Study	Randomisation and allocation process	Baseline characteristics	Blinding of participants and personnel	Incomplete outcome data	Selective reporting	Conflicts of interest
[28] Terburg et al. 2013	Low	Low	Low	Low	Low	High
[29] Nell et al. 2013	Low	Low	Low	Low	Some concern	High
[30] Chiu et al. 2014	Low	Some concern	Low	Low	Low	High
[11] Dimpfell et al. 2016 (a)	Low	Low	Low	Low	Some concern	High
[12] Dimpfell et al. 2017	Low	Low	Low	Low	Some concern	High
[31] Hoffman et al. 2020	Low	Low	Low	Low	Some concern	Some concern
[32] Reay et al. 2020	Low	Low	Low	Low	Low	High
[33] Berry et al. 2021	Low	Low	Low	Low	Some concern	Some concern

Collectively, the clinical studies discussed provide several positive findings but also unclear support for the anxiolytic, cognitive, and performance-enhancing potential of Zembrin, with most studies reporting only favourable tolerability and little to no adverse events. Several critical limitations undermine the overall strength of evidence. These include studies with small sample sizes, use of healthy participants in all studies, absence of positive controls, inconsistent reporting of baseline data, reliance on subjective self-report measures, and the absence of clinically meaningful objective outcomes in many studies. Sample sizes varied across all studies, ranging from 16 to 60 participants, where the majority used smaller sample sizes with both male and female participants. Using a small sample size increases the possibility of false positives and negatives and limits generalisability. Treatment durations ranged from a single acute dose to long-term administration over several weeks, although only two studies exceeded a month of treatment. This significantly limits data regarding the long-term efficacy, toxicity, dependence, and tolerance of Zembrin.

While preclinical studies validate promising therapeutic effects, such as serotonin reuptake inhibition and PDE4 inhibition, large variability in dosage and formulation complicates direct comparison across all studies. The use of standardised herbal extracts is critically important to ensure consistent efficacy and safety [7]. Recent publications show that the use of Zembrin as a standardised M. tortuosum extract has increased, improving the reliability of results, although, as evident from the discussion in the in vivo section, an optimal dosage of mesembrine alkaloids and/or Zembrin still needs to be established, especially considering that the clinical studies were conducted on healthy participants and that the obtained results were of no statistical relevance for the treatment of anxiety or depression. Additionally, the absence of positive controls complicates direct comparisons and introduces potential bias.

In 2016, a study was conducted in which 640 clinical studies on novel therapeuticsʼ validity and strength of evidence were evaluated based on the regulatory factors related to the FDAʼs drug approval process to assess the overall high publication rate [46]. It was concluded that 57% of the clinical studies failed to establish adequate efficacy, and 17% failed due to inadequate safety [47]. Failure can most often be attributed to poor translation from animal studies to human clinical studies. Due to the psychological and physiological differences in animal models compared with human models, preclinical studies often yield unreliable results, especially when studying a potential neuroactive compound (e.g., Zembrin), and subsequently lead to poor translation into human clinical studies [36]. For example, many drugs that are neuroactive need to penetrate the BBB efficiently to be available in high therapeutic concentrations, and it is known that the BBB of rodents is less permeable than that of humans [37]. Additionally, higher doses than those given clinically to humans are often used when conducting animal studies, creating unreliable efficacy and safety margins. This suggests that it would be beneficial to evaluate the efficacy of various administration routes in future research.

While Zembrin exhibits a favourable short-term safety profile and promising neurophysiological activity, the current clinical evidence is insufficient to make definitive conclusions regarding its therapeutic efficacy.

Conclusions and Future Research

The current evidence evaluating the therapeutic efficacy and potential of M. tortuosum and Zembrin suggests several positive findings, but careful interpretation is intricate and can lead to an inconclusive outcome regarding its main indications as an anxiolytic and antidepressant. In vivo studies showed encouraging potential indications of anxiolytic, antidepressant, analgesic, and cognitive-enhancing properties, yet these findings are substantially weakened by methodological limitations and inconsistencies. Some concerns regarding ‘promotional research’ are also evident, especially where published outcomes are incorrectly cited to appear more positive than those reported; for example, Hoffman et al. [31] cite the earlier work of Chiu et al. [30]. This is, in general, a growing concern, as demonstrated by [48], and ties in well with the well-known ‘irreproducibility’ problem in preclinical research as discussed earlier.

Clinical studies, while employing randomised, placebo-controlled designs, similarly yield inconclusive results regarding the anxiolytic, cognitive-enhancing, or mood-modulating effects of Zembrin and/or M. tortuosum. Many clinical studies are limited by small sample sizes, inconsistent placebo formulations, absence of positive controls, reliance on subjective self-report measures, and the predominant use of healthy participants rather than clinically diagnosed populations. Although favourable short-term safety profiles have been consistently reported, few studies assess long-term safety, dependence, or tolerance.

The recently published systematic review and meta-analysis by Gouhie et al. [14] reinforces these concerns, concluding that “These results indicate no clinically significant difference in anxiety between the intervention and control groups”. Their meta-analysis of four RCTs (117 participants) revealed no clinically significant differences in anxiety outcomes between M. tortuosum/Zembrin and placebo groups. Although the meta-analysis itself is limited by the number of participants (n = 117) and variability in study duration, it supports existing research suggesting that M. tortuosum/Zembrin cannot yet be considered an established anxiolytic therapy.

Collectively, available in vivo and clinical data neither definitively confirm nor disprove the therapeutic potential of M. tortuosum or Zembrin. We therefore come to a similar conclusion as Gouhie et al. [14], who concluded that “The main result obtained was inconclusive information about the use of Sceletium tortuosum in anxiety treatment, as no statical [sic] relevance indicates its efficacy. Thus, the use of this phytochemical substance as an anxiety therapy cannot be recommended by the current literature”.

The lack of consistent results and statistical relevance obtained from human clinical studies in the existing literature could be due to different factors, as mentioned previously, but also potentially to the poor bioavailability of the active components. When considering the biological differences between animals and humans, it is critical to evaluate new formulations, dosages, and different routes of administration to enhance drug delivery. Additionally, studies should explore the isolated therapeutic potential of mesembrine alkaloids to better understand their individual contributions to the efficacy and safety of M. tortuosum.

Recent in vivo pre-clinical animal studies and clinical studies seem to be prioritising the use of a standardised M. tortuosum extract formulation, namely Zembrin. This is a step in the right direction, considering that the studies discussed provide strong evidence for a good safety profile. Future in vivo studies should make use of robust chemical characterisation, larger sample sizes, blinding of researchers, inclusion of both positive and negative controls, thorough adverse event monitoring, and the use of translationally relevant models to better define the therapeutic window and optimise the clinical potential of M. tortuosum and Zembrin. Future clinical studies should prioritise larger trials incorporating standardised placebo formulations, positive controls, potentially higher doses of Zembrin, clinically validated outcomes, rigorous adverse event monitoring, and evaluations within clinically relevant populations to substantiate these preliminary findings. A clinical trial was registered in 2022 and was completed using 110 participants, but no results have yet been published (personal communication). The main objective of this study was “The aim of the proposed randomized, double-blind, placebo-controlled, parallel groups study is to assess the effects of 8 weeks supplementation with Zembrin, in 30 – 50-year-old healthy adults, on cognitive function, mood, psychological and physiological stress responses during a laboratory stressor, fatigue and sleep quality” (Trial ID: NCT05471804) [49]. Even though the results are unknown, it is a step in the right direction regarding sample size, but here again, a clinically irrelevant population was used. Only through rigorous and unbiased investigation can the therapeutic potential of M. tortuosum be reliably assessed and translated into clinical practice.

Contributorsʼ Statement

Conceptualisation: MdJ, FvdK, SvN; data curation: MdJ; formal analysis: MdJ; investigation: MdJ, FvdK; methodology: MdJ, FvdK; project administration: FvdK; software: MdJ; supervision: FvdK, SvN, JHH; validation: MdJ; writing–original draft: MdJ; writing–review and editing: FvdK, SvN, JHH. All authors have read and agreed to the published version of the manuscript.

Data availability

Data are available on request from the corresponding author.

Conflict of Interest

The authors declare to have no conflicts of interest.

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Publication History

Received: 23 September 2025

Accepted after revision: 26 January 2026

Article published online:
02 March 2026