Sceletium tortuosum: A review on its phytochemistry, pharmacokinetics, biological, pre-clinical and clinical activities.

ETHNOPHARMACOLOGICAL RELEVANCE: Sceletium tortuosum (L.) N.E.Br., the most sought after and widely researched species in the genus Sceletium is a succulent forb endemic to South Africa. Traditionally, this medicinal plant is mainly masticated or smoked and used for the relief of toothache, abdominal pain, as a mood-elevator, analgesic, hypnotic, anxiolytic, thirst and hunger suppressant, and for its intoxicating/euphoric effects. Sceletium tortuosum is currently of widespread scientific interest due to its clinical potential in treating anxiety and depression, relieving stress in healthy individuals, and enhancing cognitive functions. These pharmacological actions are attributed to its phytochemical constituents referred to as mesembrine-type alkaloids. AIM OF THE REVIEW: The aim of this review was to comprehensively summarize and critically evaluate recent research advances on the phytochemistry, pharmacokinetics, biological, pre-clinical and clinical activities of the medicinal plant S. tortuosum. Additionally, current ongoing research and future perspectives are also discussed. METHODS: All relevant scientific articles, books, MSc and Ph.D. dissertations on botany, behavioral pharmacology, traditional uses, and phytochemistry of S. tortuosum were retrieved from different databases (including Science Direct, PubMed, Google Scholar, Scopus and Web of Science). For pharmacokinetics and pharmacological effects of S. tortuosum, the focus fell on relevant publications published between 2009 and 2021. RESULTS: Twenty-five alkaloids belonging to four structural classes viz: mesembrine, Sceletium A4, joubertiamine, and tortuosamine, have been identified from S. tortuosum, of which the mesembrine class is predominant. The crude extracts and commercially available standardized extracts of S. tortuosum have displayed a wide spectrum of biological activities (e.g. antimalarial, anti-oxidant, neuromodulatory, immunomodulatory, anti-HIV, neuroprotection) in in vitro or in vivo studies. While the plant has been studied in clinical populations, this has only been in healthy subjects, so that further study in pathological states remains to be done. Nevertheless, the aforementioned studies have demonstrated that S. tortuosum has potential for enhancing cognitive function and managing anxiety and depression. CONCLUSION: As an important South African medicinal plant, S. tortuosum has garnered many research advances on its phytochemistry and biological activities over the last decade. These scientific studies have shown that S. tortuosum has various bioactivities. The findings have further established the link between the phytochemistry and pharmacological application, and support the traditional use of S. tortuosum in the indigenous medicine of South Africa.

Quantification of hordenine in a complex plant matrix by direct analysis in real time-high-resolution mass spectrometry: Application to the "plant of concern" Sceletium tortuosum.

Recently, there has been an increase in the recreational abuse of several psychoactive plants, resulting in the United Nations Office on Drugs and Crime creating a list of "plants of concern." One such material is Sceletium tortuosum and products derived from it. Regulation of these materials is challenging because of their innocuous appearance, the cumbersome sample preparation steps required to render the material into a form amenable to analysis by conventional techniques, the requirement for nuanced sample analysis protocols, and lengthy analysis times. It is demonstrated here that direct analysis in real time-high-resolution mass spectrometry (DART-HRMS) can be used to not only identify S. tortuosum material based on the detection of characteristic biomarkers including hordenine and several mesembrine alkaloids, but also quantify the amount of hordenine present. Using hordenine-d6 as an internal standard, a protocol, validated according to US Food and Drug Administration (FDA) Guidelines for the Development and Validation of Bioanalytical Methods, was devised for the quantification of the psychoactive component hordenine. The method was then applied to the quantification of hordenine in six commercially available products derived from the foliage and stems of S. tortuosum. By this method, the lower limit of quantification (LLOQ) was found to be 1 µg/ml. Observed hordenine concentrations ranged from 0.02738 to 1.071 mg of hordenine per gram of plant material. The developed technique provides an effective and quick means for the detection and quantification of hordenine in S. tortuosum, which can be extended to analysis of other hordenine-containing products.

An acute dose-ranging evaluation of the antidepressant properties of Sceletium tortuosum (Zembrin®) versus escitalopram in the Flinders Sensitive Line rat.

ETHNOPHARMACOLOGICAL RELEVANCE: Sceletium tortuosum (L.) N.E.Br. (ST) has been used by the Khoisan people of South Africa as a mood elevator. Its various pharmacological mechanisms of action suggest distinct potential as an antidepressant. Clinical studies in healthy individuals suggest beneficial effects on mood, cognition, and anxiety. AIM OF THE STUDY: To obtain a chromatographic fingerprint of a standardized extract of S. tortuosum (Zembrin®), and to evaluate the acute antidepressant-like properties of Zembrin® versus the reference antidepressant, escitalopram, in the Flinders Sensitive Line (FSL) rat, a genetic rodent model of depression. MATERIALS AND METHODS: The chemical profile of Zembrin® was determined by ultra-performance liquid chromatography-mass spectrometry (UPLC-MS) chromatogram method using alkaloid standards. Twelve saline treated FSL and six Flinders Resistant Line (FRL) control rats were used to confirm face validity of the FSL model using the forced swim test (FST). Thereafter, FSL rats (n = 10) received either 5, 10, 25, 50 or 100 mg/kg of Zembrin®, or 5, 10 or 20 mg/kg escitalopram oxalate (ESC), both via oral gavage, and subjected to the open field test (OFT) and FST. RESULTS: Four main ST alkaloids were identified and quantified in Zembrin® viz. mesembrenone, mesembrenol, mesembrine, and mesembranol (47.9%, 32%, 13.2%, and 6.8% of the total alkaloids, respectively). FSL rats showed significantly decreased swimming and climbing (coping) behaviours, and significantly increased immobility (despair), versus FRL controls. ESC 5 mg/kg and Zembrin® 25 mg/kg and 50 mg/kg showed significant dose-dependent reversal of immobility in FSL rats and variable effects on coping behaviours. Zembrin® 50 mg/kg was the most effective antidepressant dose, showing equivalence to ESC 5. CONCLUSIONS: Zembrin® (25 and 50 mg/kg) and ESC (5 mg/kg) are effective antidepressants after acute treatment in the FST, as assessed in FSL rats. Moreover, Zembrin® 50 mg/kg proved equivalent to ESC 5. Further long-term bio-behavioural studies on the antidepressant properties of Zembrin® are warranted.

Sceletium tortuosum: A review on its phytochemistry, pharmacokinetics, biological and clinical activities.

ETHNOPHARMACOLOGICAL RELEVANCE: Sceletium tortuosum (L.) N.E.Br, the most sought after and widely researched species in the genus Sceletium is a succulent forb endemic to South Africa. Traditionally, this medicinal plant is mainly masticated or smoked and used for the relief of toothache, abdominal pain, and as a mood-elevator, analgesic, hypnotic, anxiolytic, thirst and hunger suppressant, and for its intoxicating/euphoric effects. Sceletium tortuosum is currently of widespread scientific interest due to its clinical potential in treating anxiety and depression, relieving stress in healthy individuals, and enhancing cognitive functions. These pharmacological actions are attributed to its phytochemical constituents referred to as mesembrine-type alkaloids. AIM OF THE REVIEW: The aim of this review was to comprehensively summarize and critically evaluate recent research advances on the phytochemistry, pharmacokinetics, biological and clinical activities of the medicinal plant S. tortuosum. Additionally, current ongoing research and future perspectives are also discussed. METHODS: All relevant scientific articles, books, MSc and Ph.D. dissertations on botany, behavioral pharmacology, traditional uses, and phytochemistry of S. tortuosum were retrieved from different databases (including Science Direct, PubMed, Google Scholar, Scopus and Web of Science). For pharmacokinetics and pharmacological effects of S. tortuosum, the focus fell on relevant publications published between 2009 and 2021. RESULTS: Twenty-five alkaloids belonging to four structural classes viz: mesembrine, Sceletium A4, joubertiamine, and tortuosamine, have been identified from S. tortuosum, of which the mesembrine class is predominant. The crude extracts and commercially available standardized extracts of S. tortuosum have displayed a wide spectrum of biological activities (e.g. antimalarial, anti-oxidant, immunomodulatory, anti-HIV, neuroprotection, enhancement of cognitive function) in in vitro or in vivo studies. This plant has not yet been studied in a clinical population, but has potential for enhancing cognitive function, and managing anxiety and depression. CONCLUSION: As an important South African medicinal plant, S. tortuosum has garnered many research advances on its phytochemistry and biological activities over the last decade. These scientific studies have shown that S. tortuosum has various bioactivities. The findings have further established the link between the phytochemistry and pharmacological application, and support the traditional use of S. tortuosum in the indigenous medicine of South Africa.

A Chewable Cure "Kanna": Biological and Pharmaceutical Properties of Sceletium tortuosum.

Sceletium tortuosum (L.) N.E.Br. (Mesembryanthemaceae), commonly known as kanna or kougoed, is an effective indigenous medicinal plant in South Africa, specifically to the native San and Khoikhoi tribes. Today, the plant has gained strong global attraction and reputation due to its capabilities to promote a sense of well-being by relieving stress with calming effects. Historically, the plant was used by native San hunter-gatherers and Khoi people to quench their thirst, fight fatigue and for healing, social, and spiritual purposes. Various studies have revealed that extracts of the plant have numerous biological properties and isolated alkaloids of Sceletium tortuosum are currently being used as dietary supplements for medicinal purposes and food. Furthermore, current research has focused on the commercialization of the plant because of its treatment in clinical anxiety and depression, psychological and psychiatric disorders, improving mood, promoting relaxation and happiness. In addition, several studies have focused on the isolation and characterization of various beneficial bioactive compounds including alkaloids from the Sceletium tortuosum plant. Sceletium was reviewed more than a decade ago and new evidence has been published since 2008, substantiating an update on this South African botanical asset. Thus, this review provides an extensive overview of the biological and pharmaceutical properties of Sceletium tortuosum as well as the bioactive compounds with an emphasis on antimicrobial, anti-inflammatory, anti-oxidant, antidepressant, anxiolytic, and other significant biological effects. There is a need to critically evaluate the bioactivities and responsible bioactive compounds, which might assist in reinforcing and confirming the significant role of kanna in the promotion of healthy well-being in these stressful times.

Formulation of Medicated Chewing Gum Containing Sceletium tortuosum and Process Optimization Utilizing the SeDeM Diagram Expert System.

Sceletium tortuosum is one of the most promising medicinal plant species for treating anxiety and depression. Traditionally, aerial parts are chewed (masticatory herbal medicine) providing fast relief and rendering the masticatory route for delivery, ideal. This study intended formulating novel medicated chewing gum containing S. tortuosum to alleviate depression and anxiety. S. tortuosum extract was formulated into directly compressed medicated chewing gum (MCG) containing different Health-in-Gum® (HIG) bases through process optimization with the SeDeM Diagram Expert System. Physical properties of MCGs were characterized, and specialized drug release studies performed. According to the manufacturer, only HIG-03 was specifically developed for direct compression; however, the SeDeM System was successfully applied to all HIG-bases investigated. HIG-01 and HIG-04 are also considered useful in direct compression as no considerable differences in these MCG formulations' physical properties were recognized. Inclusion of a lubricant, however, is deemed essential, and MCG comprising HIG-01, most suited for direct compression. Dissolution experiments found only two alkaloids used as markers, mesembrine and mesembrenone, were released in quantifiable concentrations regardless formulation constituents. Novel directly compressed MCG-containing S. tortuosum extract was successfully formulated by which the biologically active phytochemicals of S. tortuosum can be scientifically delivered through the traditionally applied mastication method.

First Report: Diversity of Endophytic fungi Possessing Antifungal Activity Isolated from Native Kougoed (Sceletium tortuosum L.).

Forty-three (n = 43) endophytic fungi with different morphologic characteristics were from a medicinal plant Sceletium tortuosum, were utilized to investigate their antifungal effectiveness against pathogenic fungi. All fungal isolates exhibited antifungal activity against one or more pathogens in the dual culture test whereas only 33 fungal culture filtrates (77%) showed decent antifungal effect. Fusaria and Aspergillus were the dominate genus that displayed significant antifungal activity. Isolates GG02, GG09, ND15, and ND17 showed the broadest spectrum of antifungal activity. Furthermore, culture filtrate of Fusarium sp. DR08 exhibited a broad range of antifungal activity against all the pathogens. The results suggest endophytic fungi isolated from medicinal plant might be a source of novel bioactive molecules. To the best our knowledge, this is the first report on endophytic fungi isolated from native kougoed exhibiting antifungal activity against plant fungal pathogens.

Sceletium tortuosum (Zembrin® ) ameliorates experimentally induced anxiety in healthy volunteers.

OBJECTIVE: To investigate the anxiolytic properties of a standardized extract of Sceletium tortuosum (trademarked-Zembrin® ). METHODS: Two studies utilized a placebo-controlled, double-blind, between-subject experimental design to investigate the effects of a single dose of Sceletium tortuosum (25 mg, Zembrin® ) on laboratory stress/anxiety responding in 20 young healthy volunteers. To elicit feelings of stress/anxiety, participants completed 20 min of the multitasking framework in study 1 and a 5-min simulated public speaking task in study 2. Study 1 measured subjective experiences of mood at baseline, prestress induction, and poststress induction. Study 2 measured subjective experiences of anxiety and physiological indicators of stress (heart rate [HR] and galvanic skin response) at baseline, prestress induction, during stress induction, and poststress induction. RESULTS: A series of analysis of covariances (baseline entered as the covariate) revealed no treatment effect in study 1; however, study 2 revealed subjective anxiety levels to be significantly lower in the Zembrin® group at the prestress induction point and a significant interaction between treatment and time on HR. Taken together, results indicate that a single dose of Zembrin® can ameliorate laboratory stress/anxiety responding in healthy volunteers. CONCLUSION: We provide the first tentative behavioral evidence to support the anxiolytic properties of Sceletium tortuosum (25 mg Zembrin® ).

Effect of Zembrin® and four of its alkaloid constituents on electric excitability of the rat hippocampus.

ETHNOPHARMACOLOGICAL RELEVANCE: Sceletium tortuosum (Mesembryanthemaceae), a succulent plant indigenous to South Africa. is consumed in the form of teas, decoctions and tinctures and is sometimes smoked and used as snuff. In recent years, Sceletium has received a great deal of commercial interest for relieving stress in healthy people, and for treating a broad range of psychological, psychiatric and inflammatory conditions. MATERIAL AND METHODS: The whole extract (Zembrin®) was tested ex vivo in the hippocampus slice preparation after one week of daily oral administration of 5 and 10 mg/kg. Four alkaloids - mesembrine, mesembranol, mesembrenol and mesembrenone - were tested directly in vitro. All four were also tested in the presence of different glutamate receptor agonists. RESULTS: Zembrin® ex vivo as well as all alkaloids in vitro attenuated the amplitude of the population spike during electric stimulation as single shock as well as theta burst stimulation. Only Mesembranol and Mesembrenol having a hydroxyl group at position C6 instead of carbonyl group as in mesembrine and mesembrenone acted by attenuation of AMPA receptor mediated transmission as documented for the whole extract. DISCUSSION: The current experimental series revealed a new physiological effect of Zembrin® on the electric activity of the hippocampus. Attenuation of AMPA mediated transmission has been related to successful adjunctive treatment of epileptic patients. Administered doses of 5 and 10 mg/kg are in line with a dosage of 50 mg/subject as tested clinically. CONCLUSION: We have discovered a new structure activity relationship for Sceletium alkaloids. Since attenuation of AMPA mediated transmission has been related to successful adjunctive treatment of epileptic patients), Mesembrenol and Mesembranol may serve as new chemical leads for the development of new drugs for the treatment of epilepsy.

Immunomodulatory effects of Sceletium tortuosum (Trimesemine™) elucidated in vitro: Implications for chronic disease.

ETHNOPHARMACOLOGICAL RELEVANCE: Sceletium tortuosum, among other Sceletium species, was traditionally used by the Khoisan people of Southern Africa for relief of pain-related ailments. However, the commercial availability of this supplement has greatly expanded due to anecdotal claims of its mood-elevating and anxiolytic properties. Unrelated research has elucidated a significant link between cytokines and the mediation of depression. Therefore, the effect of Sceletium supplementation on immune cell functionality is of interest, since the efficacy of potential depression treatments could, at least in part, rely on downregulation of pro-inflammatory signalling. AIM OF THE STUDY: The current study evaluated the immunomodulatory effects of a Sceletium extract, both basally and in the context of acute endotoxin stimulation. MATERIALS AND METHODS: Primary human monocytes were supplemented with either a 0.01mg/ml or 1mg/ml Sceletium extract dose, with or without E. coli LPS stimulation in vitro, for 24h. Mitochondrial viability, as an indirect measure of cytotoxicity, and cytokine release in response to the treatment intervention were assessed. RESULTS: Sceletium extract treatment was associated with increased mitochondrial viability, as well as up-regulated IL-10 release under basal conditions. LPS exposure significantly decreased mitochondrial viability, but this was prevented completely under Sceletium-treated conditions. The acute inflammatory response to LPS stimulation was not negatively affected. Sceletium treatment conferred most significant effects at a dose of 0.01mg/ml. CONCLUSIONS: Sceletium exerts significant cytoprotective effects in the setting of endotoxin stimulation. Cytokine assessment indicated that Sceletium possesses mild anti-inflammatory properties, but does not hinder the mounting of an adequate immune response to acute immune challenge. These findings indicate that Sceletium may be beneficial for the attenuation of cytokine-induced depression, as well as in systemic low-grade inflammation.

Mesembrine alkaloids: Review of their occurrence, chemistry, and pharmacology.

ETHNOPHARMACOLOGICAL RELEVANCE: Mesembrine alkaloids are considered to be the primary active constituents of the South African medicinal plant Sceletium tortuosum (L.) N.E.Br. (Aizoaceae), and it is used as the dried or fermented aerial material from the plant, which is known as kanna (aka, channa, kougoed). Traditional regional use ranged from relieving thirst, mild analgesia, and alteration of mood. Current interest has focused primarily on the antidepressant action of preparations based on the plant and commercialization is expanding the recognition and availability of these preparations. MATERIALS AND METHODS: Searches for the keywords "Sceletium or mesembrine" were performed in "PubMed-NCBI", "Chemical Abstracts SciFinder" and "Thomson Reuters Web of Science" databases in addition to the inclusion of references cited within prior reviews and scientific reports. Additionally the "SciFinder" database was searched using 3a-phenyl-cis-octahydroindole in the SciFinder Substructure Module (SSM). Plant taxonomy was validated by the database "The Plant List". RESULTS: This review focuses on the chemistry, analysis, and pharmacology of the mesembrine alkaloids. Despite a long history of medicinal used and research investigation, there has been a renewed interest in the pharmacological properties of the mesembrine alkaloids and much of the pharmacology has only recently been published. The two major active alkaloids mesembrine and mesembrenone are still in the process of being more fully characterized pharmacologically. They are serotonin reuptake inhibitors, which provides a rationale for the plant's traditional use as an antidepressant, but other actions are beginning to appear in the literature. Additionally, mesembrenone has reasonably potent PDE4 inhibitory activity. This review intends to provide an overview of the available literature, summarize the current findings, and put them in perspective with earlier studies and reviews.

The effects of Sceletium tortuosum (L.) N.E. Br. extract fraction in the chick anxiety-depression model.

ETHNOPHARMACOLOGICAL RELEVANCE: Sceletium tortuosum (L.) N.E. Br. has been reported to elevate mood, reduce anxiety and stress and alleviate pain. AIM OF STUDY: This study sought to examine the effects of an S. tortuosum alkaloid enriched fraction in the chick anxiety-depression model, a model that shows high predictive validity as a pharmacological screening assay. MATERIAL AND METHODS: Socially-raised male Silver Laced Wyandotte chicks (4-6 days old) were given IP vehicle, imipramine (10mg/kg), or S. tortuosum fraction (10, 20, 30mg/kg in Exp. 1 or 50, 75, 100mg/kg in Exp. 2) 15min prior to a 60min isolation test period in which distress vocalizations (DVoc) were continuously recorded. RESULTS: Vehicle chicks displayed high DVoc rates in the anxiety phase (first 3min). DVoc rates declined about 50% (i.e., behavioral despair) in the depression phase (30-60min). S. tortuosum fraction at 75 and 100mg/kg decreased DVoc rates during the anxiety phase indicative of an anxiolytic effect. Imipramine, but not S. tortuosum groups, increased DVoc rates in the depression phase indicative of an antidepressant effect. CONCLUSIONS: The findings suggest that an alkaloid enriched S. tortuosum fraction may benefit some forms of stress-related disorders.

Direct analysis in real time high resolution mass spectrometry as a tool for rapid characterization of mind-altering plant materials and revelation of supplement adulteration--The case of Kanna.

We demonstrate the utility of direct analysis in real time ionization coupled with high resolution time-of-flight mass spectrometry (DART-HRTOFMS) in revealing the adulteration of commercially available Sceletium tortuosum, a mind-altering plant-based drug commonly known as Kanna. Accurate masses consistent with alkaloids previously isolated from S. tortuosum plant material enabled identification of the products as Kanna, and in-source collision-induced dissociation (CID) confirmed the presence of one of these alkaloids, hordenine, while simultaneously revealing the presence of an adulterant. The stimulant ephedrine, which has been banned in herbal products and supplements, was confirmed to be present in a sample through the use of in-source CID. High-throughput DART-HRTOFMS was shown to be a powerful tool to not only screen plant-based drugs of abuse for psychotropic alkaloids, but also to reveal the presence of scheduled substances and adulterants.

Electropharmacogram of Sceletium tortuosum extract based on spectral local field power in conscious freely moving rats.

ETHNOPHARMACOLOGICAL RELEVANCE: The endemic succulent South African plant, Sceletium tortuosum (L.) N.E. Br. (synonym Mesembryanthemum tortuosum L.), of the family Mesembryathemaceae, has an ancient oral tradition history of use by San and Khoikhoi people as an integral part of the indigenous culture and materia medica. A special standardized extract of Sceletium tortuosum (Zembrin®) has been developed and tested pre-clinically in rats, and clinically in healthy subjects. AIM OF THE STUDY: The present investigation aimed at the construction of electropharmacograms of Zembrin® in the presence of three dosages (2.5, 5.0 and 10.0 mg/kg), and comparative electropharmacograms and discriminatory analyses for other herbal extracts, citicoline and rolipram. MATERIAL AND METHODS: Seventeen adult Fischer rats were each implanted with a set consisting of four bipolar concentric steel electrodes fixed by dental cement and three screws driven into the scalp. After two weeks of recovery from surgery the animals were adapted to oral administration by gavage and to experimental conditions (45 min pre-drug period and 5h of recording after a rest of 5 min for calming down). Data were transmitted wirelessly and processed using a Fast Fourier Transformation (FFT). Spectral power was evaluated for 8 frequency ranges, namely delta, theta, alpha1, alpha2, beta1a, beta1b, beta2 and gamma power. RESULTS: Zembrin® dose dependently attenuated all frequency ranges, to varying degrees. The most prominent was the statistically significant reduction in alpha2 and beta1a waves, correlated with activation of the dopaminergic and glutamatergic transmitter systems respectively. This feature is common to all synthetic and herbal stimulants tested to date. The second strongest effects were reduction in both the delta and the theta frequency ranges, correlated with changes in the cholinergic and norepinephrine systems respectively, a pattern seen in preparations prescribed for neurodegenerative diseases. Theta wave reduction in common with the delta, alpha2 and beta1 attenuation has been noted for analgesic drugs. Attenuation of alpha1 waves emerged during the highest dosage in all brain areas, a feature seen in all antidepressants. DISCUSSION: The electropharmacogram of Zembrin® was compared to the electropharmacograms of herbal extracts archived in our database. Extracts of Oenothera biennis and Cimicifuga racemosa gave a very similar electropharmacograms to that of Zembrin®, and extracts of Ginkgo biloba and Rhodiola rosea gave rather similar electropharmacograms to Zembrin®. Linear discriminant analysis confirmed these similarities and demonstrated that all three dosages of Zembrin® plotted in close neighbourhood to each other. Citocoline, a synthetic compound originally developed for cognitive enhancement, had a similar electropharmacogram to Zembrin®. Similarity to the electropharmacograms of the synthetic phosphodiesterase-4 inhibitor, rolipram, suggests Zembrin® has antidepressant and cognitive function enhancing potential. CONCLUSION: The combined results from the electropharmacograms and comparative discriminatory analyses suggest that Zembrin® has dose dependent activity, with potential applications as a cognitive function enhancer, as an antidepressant, and as an analgesic.

Modulation of glucocorticoid, mineralocorticoid and androgen production in H295 cells by Trimesemine™, a mesembrine-rich Sceletium extract.

ETHNOPHARMACOLOGICAL RELEVANCE: Stress-related illnesses rate among the most prevalent non-fatal diseases globally. With the global trend for consumer bias towards natural medicine, the Sceletium plant has become more prominent in the field of natural products. Although potentially useful effects of Sceletium tortuosum on the central nervous system have been reported, limited data is available on effects of the plant in the peripheral compartment. AIM OF THE STUDY: The current study aimed to elucidate the effect(s) of a Sceletium extract (TRI) rich in mesembrine (1% of plant extract w/w), on adrenal steroid biosynthesis. MATERIALS AND METHODS: Steroidogenesis was assessed basally and in response to stimuli (forskolin, angiotensin II, KCl), in human adrenocortical carcinoma cells (H295R). Steroid hormone levels were assessed using UPLC-MS/MS. UPLC-MS analyses of TRI identified major alkaloids Δ7-mesembrenone, mesembrenone and mesembrine. RESULTS: Highest dose TRI treatment (1 mg/ml, 34.5 µM mesembrine) increased pregnenolone and decreased 16-hydroxyprogesterone levels (both P<0.00001) in forskolin-stimulated conditions only, suggesting CYP17 enzyme inhibition. This led to significant inhibition of forskolin-associated increases in cortisol levels at the highest dose (P<0.001) and basal cortisol levels across all doses (P<0.0001). Independently of forskolin, TRI inhibited androstenedione and testosterone production across all doses (both P<0.00001), suggesting inhibition of 3ßHSD and 17ßHSD respectively. TRI decreased both the angiotensin II- (P<0.05) and forskolin-induced (P<0.0001) increases in aldosterone production. CONCLUSIONS: Our data suggest potentially beneficial effects of TRI in the context of stress and hypertension. These should be further investigated in a whole organism model, while the effects on the androgenic pathway should also be further elucidated.

High-mesembrine Sceletium extract (Trimesemine™) is a monoamine releasing agent, rather than only a selective serotonin reuptake inhibitor.

ETHNOPHARMACOLOGICAL RELEVANCE: Extracts from and alkaloids contained in plants in the genus Sceletium have been reported to inhibit ligand binding to serotonin transporter. From this, the conclusion was made that Sceletium products act as selective serotonin-reuptake inhibitors. However, other mechanisms which may similarly result in the anxiolytic or anti-depressant effect ascribed to Sceletium, such as monoamine release, have not been investigated. AIMS OF THE STUDY: The current study investigated simultaneously and at two consecutive time points, the effect of high-mesembrine Sceletium extract on both monoamine release and serotonin reuptake into both human astrocytes and mouse hippocampal neurons, as well as potential inhibitory effects on relevant enzyme activities. MATERIALS AND METHODS: Human astrocytes and mouse hippocampal cells were treated with citalopram or Sceletium extract for 15 and 30min, after which protein expression levels of serotonin transporter (SERT) and vesicular monoamine transporter-2 (VAMT-2) was assessed using fluorescent immunocytochemistry and digital image analysis. Efficacy of inhibition of acetylcholinesterase (AChE) and monoamine oxidate-A (MAO-A) activity were assessed using the Ellman and Olsen methods (and appropriate controls) respectively. RESULTS: We report the first investigation of mechanism of action of Sceletium extract in the context of serotonin transport, release and reuptake in a cellular model. Cell viability was not affected by Sceletium treatment. High-mesembrine Sceletium extract down-regulated SERT expression similarly to citalopram. In addition, VMAT-2 was upregulated significantly in response to Sceletium treatment. The extract showed only relatively mild inhibition of AChE and MAO-A. CONCLUSIONS: We conclude that the serotonin reuptake inhibition activity ascribed to the Sceletium plant, is a secondary function to the monoamine-releasing activity of high-mesembrine Sceletium extract (Trimesemine(TM)).

A toxicological safety assessment of a standardized extract of Sceletium tortuosum (Zembrin®) in rats.

A well-characterized standardized hydroethanolic extract of a traditionally recognized mak (mild) variety of Sceletium tortuosum, a South African plant with a long history of traditional ingestion, is marketed under the trade name Zembrin(®) as an ingredient for use in functional foods and dietary supplements. It is standardized to contain 0.35-0.45% total alkaloids (mesembrenone and mesembrenol ≥60%, and mesembrine <20%). A 14-day repeated oral toxicity study was conducted at 0, 250, 750, 2500, and 5000 mg/kg bw/day. A 90-day subchronic repeated oral toxicity study was conducted at 0, 100, 300, 450, and 600 mg/kg bw/day. Because S. tortuosum has a long history of human use for relieving stress and calming, a functional observation battery, including spontaneous locomotor activity measured using LabMaster ActiMot light-beam frames system, was employed. Several parameters, such as locomotion, rearing behavior, spatial parameters, and turning behavior were investigated in the final week of the study. No mortality or treatment-related adverse effects were observed in male or female Crl:(WI)BR Wistar rats in the 14- or 90-day studies. In the 14- and 90-day studies, the NOAELs were concluded as 5000 and 600 mg/kg bw/d, respectively, the highest dose groups tested.

Effects of Sceletium tortuosum in rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Broad historical and current uses in addition to diverse activity on CNS targets may make Sceletium tortuosum a useful therapeutic in a variety of clinical settings. This study sought to more broadly characterize activity of Sceletium tortuosum and mesembrine in a number of common, rodent-based assays that model nociception, depression, anxiety, ataxia, and abuse liability. MATERIALS AND METHODS: Male Sprague-Dawley were administered Sceletium tortuosum extract products and behavioral responses were evaluated in the conditioned place preference (CPP), hot plate, forced swim, elevated plus, and rotarod tests. RESULTS AND CONCLUSIONS: Sceletium tortuosum does not cause preference or aversion in CPP. Mesembrine appears to have analgesic properties without abuse liabilities or ataxia. The Sceletium tortuosum fraction has antidepressant properties but does produce ataxia. The ataxia may limit its usefulness as an antidepressant unless the antidepressant activity is associated with one constituent and the ataxia is associated with a separate constituent.

A novel approach in herbal quality control using hyperspectral imaging: discriminating between Sceletium tortuosum and Sceletium crassicaule.

INTRODUCTION: Sceletium tortuosum is the most sought after species of the genus Sceletium and is commonly included in commercial products for the treatment of psychiatric conditions and neurodegenerative diseases. However, this species exhibits several morphological and phytochemical similarities to S. crassicaule. OBJECTIVES: The aim of this investigation was to use ultrahigh-performance liquid chromatography (UPLC) and hyperspectral imaging, in combination with chemometrics, to distinguish between S. tortuosum and S. crassicaule, and to accurately predict the identity of specimens of both species. METHODS: Chromatographic profiles of S. tortuosum and S. crassicaule specimens were obtained using UPLC with photodiode array detection. A SisuChema near infrared hyperspectral imaging camera was used for acquiring images of the specimens and the data was processed using chemometric computations. RESULTS: Chromatographic data for the specimens revealed that both species produce the psychoactive alkaloids that are used as quality control biomarkers. Principal component analysis of the hyperspectral image of reference specimens for the two species yielded two distinct clusters, the one representing S. tortuosum and the other representing S. crassicaule. A partial least squares discriminant analysis model correctly predicted the identity of an external dataset consisting of S. tortuosum or S. crassicaule samples with high accuracy (>94%). CONCLUSIONS: A combination of hyperspectral imaging and chemometrics offers several advantages over conventional chromatographic profiling when used to distinguish S. tortuosum from S. crassicaule. In addition, the constructed chemometric model can reliably predict the identity of samples of both species from an external dataset.