Skeletons in the closet? Using a bibliometric lens to visualise phytochemical and pharmacological activities linked to Sceletium, a mood enhancer.

Plants from the Sceletium genus (Aizoaceae) have been traditionally used for millennia by the Khoe and Khoen people in southern Africa, as an appetite suppressant as well as a mood elevator. In more recent times, this mood-elevating activity has been commercialised in the South African natural products industry for the treatment of anxiety and depression, with several products available both locally and abroad. Research on this species has seen rapid growth with advancements in analytical and pharmacological tools, in an effort to understand the composition and biological activity. The Web of Science (WoS) database was searched for articles related to 'Sceletium' and 'Mesembrine'. These data were additionally analysed by bibliometric software (VOSviewer) to generate term maps and author associations. The thematic areas with the most citations were South African Traditional Medicine for mental health (110) and anxiolytic agents (75). Pioneer studies in the genus focused on chemical structural isolation, purification, and characterisation and techniques such as thin layer chromatography, liquid chromatography (HPLC, UPLC, and more recently, LC-MS), gas chromatography mass spectrometry (GC-MS), and nuclear magnetic resonance (NMR) to study mesembrine alkaloids. Different laboratories have used a diverse range of extraction and preanalytical methods that became routinely favoured in the analysis of the main metabolites (mesembrine, mesembranol, mesembranone, and Sceletium A4) in their respective experimental settings. In contrast with previous reviews, this paper identified gaps in the research field, being a lack of toxicology assays, a deficit of clinical assessments, too few bioavailability studies, and little to no investigation into the minor alkaloid groups found in Sceletium. Future studies are likely to see innovations in analytical techniques like leaf spray mass spectrometry and direct analysis in real-time ionisation coupled with high-resolution time-of-flight mass spectrometry (DART-HR-TOF-MS) for rapid alkaloid identification and quality control purposes. While S. tortuosum has been the primary focus, studying other Sceletium species may aid in establishing chemotaxonomic relationships and addressing challenges with species misidentification. This research can benefit the nutraceutical industry and conservation efforts for the entire genus. At present, little to no pharmacological information is available in terms of the molecular physiological effects of mesembrine alkaloids in medical clinical settings. Research in these fields is expected to increase due to the growing interest in S. tortuosum as a herbal supplement and the potential development of mesembrine alkaloids into pharmaceutical drugs.

Corrigendum: Mass spectrometry metabolomics and feature-based molecular networking reveals population-specific chemistry in some species of the Sceletium genus.

[This corrects the article DOI: 10.3389/fnut.2022.819753.].

Mass Spectrometry Metabolomics and Feature-Based Molecular Networking Reveals Population-Specific Chemistry in Some Species of the Sceletium Genus.

The Sceletium genus has been of medicinal importance in southern Africa for millennia and Sceletium tortuosum (Aizoaceae), one of eight species in the genus has gained pharmaceutical importance as an anxiolytic and anti-depressant due to the presence of mesembrine alkaloids. S. tortuosum is used for the manufacture of herbal teas, dietary supplements and other phytopharmaceutical products. This study aimed to provide a metabolomic characterization of S. tortuosum and its sister species as these are not easy to distinguish using morphology alone. Plant samples were thus collected from various locations in the succulent Karoo (South Africa) and analyzed through liquid chromatography-mass spectrometry (LC-MS), using MSE fragmentation as a putative tool for chemical identities. Metabolomics-based analyses in combination with molecular networking were able to distinguish between the four species of Sceletium based on the presence of 4-(3,4-dimethyoxyphenyl)-4-[2-acetylmethlamino)ethyl]cyclohexanone (m/z 334.2020; RT 6.60 min), mesembrine (m/z 290.1757; RT 5.10 min) and 4'-O-demethylmesembrenol (m/z 276.1597; RT 4.17 min). Metabolomic profiles varied according to the different localities and metabolites occurred at variable quantitative levels in Sceletium ecotypes. Molecular networking provided the added advantage of being able to observe mesembrine alkaloid isomers and coeluting metabolites (from the joubertiamine group) that were difficult to discern without this application. By combining high-throughput metabolomics together with global and feature based-molecular networking, a powerful metabolite profiling platform that is able to discern chemical patterns within and between populations was established. These techniques were able to reveal chemotaxonomic relationships and allowed for the discovery of chemical markers that may be used as part of monitoring protocols during the manufacture of phytopharmaceutical and dietary products based on Sceletium.

Reverse ethnopharmacology and drug discovery.

ETHNOPHARMACOLOGICAL RELEVANCE: Ethnopharmacological investigations of traditional medicines have made significant contributions to plant-derived drugs, as well as the advancement of pharmacology. Drug discovery from medicinal flora is more complex than generally acknowledged because plants are applied for different therapeutic indications within and across cultures. Therefore we propose the concept of "reverse ethnopharmacology" and compare biomedical uses of plant taxa with their ethnomedicinal and popular uses and test the effect of these on the probability of finding biomedical and specifically anticancer drugs. MATERIALS AND METHODS: For this analysis we use data on taxonomy and medical indications of plant derived biomedical drugs, clinical trial, and preclinical trial drug candidates published by Zhu et al. (2011) and compare their therapeutic indications with their ethnomedicinal and popular uses as reported in the NAPRALERT® database. Specifically, we test for increase or decrease of the probability of finding anticancer drugs based on ethnomedicinal and popular reports with Bayesian logistic regression analyses. RESULTS: Anticancer therapy resulted as the most frequent biomedicinal indication of the therapeutics derived from the 225 drug producing higher plant taxa and showed an association with ethnomedicinal and popular uses in women's medicine, which was also the most important popular use-category. Popular remedies for dysmenorrhoea, and uses as emmenagogues, abortifacients and contraceptives showed a positive effect on the probability of finding anticancer drugs. Another positive effect on the probability of discovering anticancer therapeutics was estimated for popular herbal drugs associated with the therapy of viral and bacterial infections, while the highest effect was found for popular remedies used to treat cancer symptoms. However, this latter effect seems to be influenced by the feedback loop and divulgence of biomedical knowledge on the popular level. CONCLUSION: We introduce the concept of reverse ethnopharmacology and show that it is possible to estimate the probability of finding biomedical drugs based on ethnomedicinal uses. The detected associations confirm the classical ethnopharmacological approach where a popular remedy for disease category X results in a biomedical drug for disease category X but does also point out the existence of cross-over relationships where popular remedies for disease category X result in biomedical therapeutics for disease category Y (Zhu et al., 2011).

Antiinflammatory and neurological activity of pyrithione and related sulfur-containing pyridine N-oxides from Persian shallot (Allium stipitatum).

ETHNOPHARMACOLOGICAL RELEVANCE: Persian shallot (Allium stipitatum) is a bulbous plant native to Turkey, Iran and Central Asia. It is frequently used in folk medicine for the treatment of a variety of disorders, including inflammation and stress. Antiinflammatory and neurological activities of pyrithione and four related sulfur-containing pyridine N-oxides which are prominent constituents of Allium stipitatum were tested. METHODS: The antiinflammatory activity was tested by the ability of the compounds to inhibit cyclooxygenase (COX-1 and COX-2), whereas the neurological activities were evaluated by assessing the compounds ability to inhibit monoamine oxidase-A (MAO-A) and acetylcholinesterase (AChE). The compounds׳ affinity for the serotonin transport protein (SERT) and the GABAA-benzodiazepine receptor were also investigated. RESULTS: 2-[(Methylthio)methyldithio]pyridine N-oxide showed very high antiinflammatory effects which are comparable with those of common pharmaceuticals (IC50 of 7.8 and 15.4 µM for COX-1 and COX-2, respectively). On the other hand, neurological activities of the compounds were rather modest. Some compounds moderately inhibited AChE (IC50 of 104-1041 µM) and MAO-A (IC50 of 98-241 µM) and exhibited an affinity for the SERT and GABAA-benzodiazepine receptor. CONCLUSIONS: Our findings may help to rationalize the wide use of Persian shallot for the treatment of inflammatory disorders.

Can phylogeny predict chemical diversity and potential medicinal activity of plants? A case study of Amaryllidaceae.

BACKGROUND: During evolution, plants and other organisms have developed a diversity of chemical defences, leading to the evolution of various groups of specialized metabolites selected for their endogenous biological function. A correlation between phylogeny and biosynthetic pathways could offer a predictive approach enabling more efficient selection of plants for the development of traditional medicine and lead discovery. However, this relationship has rarely been rigorously tested and the potential predictive power is consequently unknown. RESULTS: We produced a phylogenetic hypothesis for the medicinally important plant subfamily Amaryllidoideae (Amaryllidaceae) based on parsimony and Bayesian analysis of nuclear, plastid, and mitochondrial DNA sequences of over 100 species. We tested if alkaloid diversity and activity in bioassays related to the central nervous system are significantly correlated with phylogeny and found evidence for a significant phylogenetic signal in these traits, although the effect is not strong. CONCLUSIONS: Several genera are non-monophyletic emphasizing the importance of using phylogeny for interpretation of character distribution. Alkaloid diversity and in vitro inhibition of acetylcholinesterase (AChE) and binding to the serotonin reuptake transporter (SERT) are significantly correlated with phylogeny. This has implications for the use of phylogenies to interpret chemical evolution and biosynthetic pathways, to select candidate taxa for lead discovery, and to make recommendations for policies regarding traditional use and conservation priorities.

South African plants used in traditional medicine to treat epilepsy have an antagonistic effect on NMDA receptor currents.

ETHNOPHARMACOLOGICAL RELEVANCE: Several Searsia species (Anacardiaceae), including Searsia dentata and Searsia pyroides, are used in South Africa traditional medicine to treat epilepsy. Ethanol leaf extracts of these plants have been shown to act as possible antagonists of N-methyl-d-aspartate (NMDA)-type glutamate receptors. MATERIALS AND METHODS: Leaf material of three Searsia species were collected from the Botanical Garden at the University of KwaZulu-Natal, Pietermaritzburg; dried and extracted with ethanol in an ultrasound bath. Filtered and dried extracts were resuspended in DMSO (100mg/ml) and diluted in the recording solution. The effect of Searsia dentata, Searsia pyroides and Searsia glauca extracts was investigated in dissociated cerebellar granule cells (CGCs) from 8-day-old rats and in transiently transfected HEK (human embryonic kidney) 293 cells (HEK), expressing either NR1a/NR2A or NR1a/NR2B receptors. In both systems we measured whole-cell currents elicited by 0.5mM NMDA (CGCs) or 50 µM glutamic acid (HEK) at -60 mV in 0Mg and 30 µM glycine and NMDA driven Ca influx in Fura2-loaded CGC. RESULTS: Searsia dentata and Searsia pyroides ethanol extracts caused a dose-dependent decrease of NR current with ED(50) close to 0.03 mg/ml in CGC and a similar inhibition (80% with 1mg/ml) in HEK cells, while Searsia glauca was much less effective. The inhibition was dependent on time of incubation and slightly favored by opening of the NR channel. It was hardly reversible during the recording time, but was not caused by accelerated run-down or by interaction with the modulatory redox site. Searsia pyroides ethanol extract also depressed the NMDA stimulated increase in intracellular Ca. CONCLUSIONS: The data confirm the specificity of Searsia dentata and Searsia pyroides and justify their use in traditional medicine. These plants may combine one or more γ-aminobutyric acid (GABA)(A) agonists with one or more NMDA antagonists, thus representing an efficient treatment for epilepsy.

Anticonvulsant effects of Searsia dentata (Anacardiaceae) leaf extract in rats.

Searsia species are used in South Africa to treat epilepsy. Previous studies have demonstrated an in vitro N-methyl-D-aspartic acid (NMDA) receptor antagonistic effect of the ethanolic leaf extract. The aim of this study was to evaluate the potential anticonvulsant properties of the ethanolic extract of S. dentata in various animal models of epilepsy. The extract was submitted to a screening in anticonvulsant assays including NMDA-, kainic acid (KA)-, pentylenetetrazol (PTZ)- and bicuculline (BIC)-induced seizures in rats. The extract protected 47% of the PN 18 Wistar pups (postnatal day 18, date of birth PN 0) (p < 0.05, n > 10) against NMDA-induced seizures and significantly delayed the onset of PTZ-induced seizures (p < 0.05, n > 8) at a dose of 250 mg/kg. A dose optimum was detected at 500 mg/kg for protection against KA-(63% protection, p < 0.05, n > 8) and BIC-induced seizures (50% protection, p < 0.05, n > 8) in young adult and PN 18 rats, respectively. The ethanolic extract of S. dentata showed anticonvulsive properties in several models of epilepsy. These results are compatible with previous findings of NMDA receptor antagonism. Due to the complex composition of the extract, the effect might be caused by more than one compound.

Amides from Piper capense with CNS activity - a preliminary SAR analysis.

Piper capense L.f. (Piperaceae) is used traditionally in South Africa as a sleep inducing remedy. Bioassay-guided fractionation of the roots of P. capense led to the isolation of piperine (1) and 4,5-dihydropiperine (2), which showed moderate affinity for the benzodiazepine site on the GABA(A) receptor (IC(50) values of 1.2 mM and 1.0 mM, respectively). The present study suggests that strict structural properties of the amides are essential for affinity. Taken together, these observations suggest that the carbon chain must contain not less than four carbons, and that a conjugated double bond, adjacent to the amide group, is necessary for binding to the receptor and that the amine part should be bulky.

Effects of South African traditional medicine in animal models for depression.

ETHNOPHARMACOLOGICAL RELEVANCE: The four South African medicinal plants Agapanthus campanulatus (AC), Boophone distica (BD), Mondia whitei (MW) and Xysmalobium undulatum (XU) are used in traditional medicine to treat depression. AIM: To evaluate the effect of ethanolic extracts of the plants in models for depression. MATERIALS AND METHODS: The extracts were screened for affinity for the serotonin transporter (SERT) in the [(3)H]-citalopram-binding assay. The inhibitory potency of the extracts towards the SERT, the noradrenalin transporter (NAT) and the dopamine transporter (DAT) were determined in a functional uptake inhibition assay. Antidepressant-like effects of the extracts were investigated using the tail suspension test (TST) and the forced swim test in both rats (rFST) and mice (mFST). RESULTS: All four plants showed affinity for SERT in the binding assay. AC and BD showed functional inhibition of SERT, NAT and DAT, MW affected SERT while XU showed no effect. BD showed significant effect in the TST and in the mFST/rFST, AC showed significant effect in mFST, MW showed significant effect in the rFST and XU showed significant effect in the mFST. CONCLUSION: In this study we have demonstrated the antidepressant activity of four South African medicinal plants in vitro and in vivo, supporting their rational use in traditional medicine.

Review on plants with CNS-effects used in traditional South African medicine against mental diseases.

The majority of the population in South Africa use traditional health care to treat various mental conditions. In this review, we present ethnobotanical information on plants used by the traditional healers in South Africa to treat mental illnesses, specifically epilepsy, depression, age-related dementia and debilitative mental disorders. Details of the recent scientific studies conducted on some of these plants are reviewed. Extracts of Searsia chirindensis, Cotelydon orbiculata and Leonotis leonurus have shown in vivo anticonvulsant activity. Extracts from Searsia dentata and Searsia pyroides showed spontaneous epileptiform discharge in mouse cortical slices, and acted as NMDA-receptor antagonists. Apigenin, amentoflavone and agathisflavone with affinity to the benzodiazepine site on the GABA(A)-receptor were isolated from Searsia pyroides. Naringenin with affinity to the GABA(A)-benzodiazepine receptor was isolated from Mentha aquatica. Agapanthus campanulatus, Boophone disticha, Mondia whitei and Xysmalobium undulatum exhibited antidepressant-like activity in three in vivo models for depression. Amaryllidaceae alkaloids with activity to the serotonin transporter were isolated from Boophone disticha. The alkaloid mesembrine, which act as a serotonin reuptake inhibitor, was isolated from Sceletium tortuosum. Investigations of plants used to treat age-related dementia and debilitative mental disorders lead to the isolation of a number of Amaryllidaceae alkaloids with acetylcholinesterase inhibitory activity from Boophone disticha and Crinum species. Extracts of Mentha aquatica, Gasteria croucheri, Ruta graveolens and Scotia brachypetala inhibited MAO-B. Naringenin was isolated from Mentha aquatica as a MAO inhibitor. Only a small number of the more than 300 southern African plant species reported to treat or affect the CNS have been scientifically evaluated. Very few of the active compounds have been isolated and identified.

Isolation of the MAO-inhibitor naringenin from Mentha aquatica L.

AIMS OF THE STUDY: To isolate the compound(s) responsible for the MAO-inhibitory activity. MATERIALS AND METHODS: Six extracts of varying polarity of Mentha aquatica L. were tested in a photometric peroxidase linked MAO bioassay. The 70% ethanol extract had highest inhibitory activity. (S)-Naringenin was isolated from the extract by bioassay guided fractionation on VLC and preparative TLC. The structure of the compound was determined by (1)H, (13)C and (13)C-DEPT NMR and optical rotation. RESULTS: The IC(50) values for MAO inhibition by naringenin were 342+/-33 microM for the rat liver mitochondrial fraction, 955+/-129 microM for MAO-A and 288+/-18 microM for MAO-B. CONCLUSIONS: The content of naringenin in Mentha aquatica might explain its use in traditional medicine for depression-like conditions.

The polyacetylene falcarindiol with COX-1 activity isolated from Aegopodium podagraria L.

Extracts of Aegopodium podagraria L. were screened in vitro for cyclooxygenase-1 (COX-1) inhibitory activity. The isolation of the active compound falcarindiol was achieved by bioassay-guided fractionation. The identification of the active compound was confirmed by (1)H NMR and (13)C NMR. The IC(50)-value of falcarindiol was 0.3 microM in the COX-1 assay. A quantitative determination of the seasonal variation in the content of falcarindiol in different plant parts was carried out by HPLC analysis. The flowers from Aegopodium podagraria collected in June 2006 had the highest concentration of falcarindiol (88 mg/g plant material).

Inhibition of [3H]citalopram binding to the rat brain serotonin transporter by Amaryllidaceae alkaloids.

Twenty-one Amaryllidaceae alkaloids isolated from different Amaryllidaceae species were investigated for their affinity to the serotonin reuptake transport protein and for GABA(A)-benzodiazepine receptor binding. Cherylline (21), crinamine (7), crinine (1), epibuphanisine (2), epivittatine (6), maritidine (11), O-methylmaritidine (12), powelline (3), 1-O-acetyllycorine (18) and tazettine ( 13) showed affinity to the serotonin reuptake transport protein. Cherylline (21) and epivittatine (6) yielded the highest activity among the group. No GABA(A)-benzodiazepine receptor binding activity was exhibited by the alkaloids tested.

Biflavones from Rhus species with affinity for the GABA(A)/benzodiazepine receptor.

In South Africa Rhus pyroides is traditionally used in the treatment of epilepsy. In the present study two biflavonoids with activity in the (3)H-Ro 15-1788 (flumazenil) binding assay were isolated by high pressure liquid chromatography (HPLC) fractionation of the ethanol extract of the leaves from Rhus pyroides. The structures of the two biflavonoids were elucidated by nuclear magnetic resonance spectroscopy (NMR) to be agathisflavone and amentoflavone. Agathisflavone and amentoflavone competitively inhibited the binding of (3)H-Ro 15-1788 with a K(i) of 28 and 37 nM, respectively. Extracts of Rhus dentata and Rhus pentheri were not as active as the extract from Rhus pyroides; both were found to contain apigenin and agathisflavone. The monomer apigenin, agathisflavone and amentoflavone were fitted into a pharmacophore model for ligands binding to the GABA(A) receptor benzodiazepine site. This reflected the affinities of the compounds in the [(3)H]-flumazenil binding assay.

Activity of traditional South African sedative and potentially CNS-acting plants in the GABA-benzodiazepine receptor assay.

Several plants are traditionally used to treat mental diseases in South Africa. Forty-six ethanol extracts from 35 species, both indigenous and exotic that are traditionally used predominantly as sedatives or to treat various CNS-related ailments were tested in the GABAA-benzodiazepine receptor-binding assay. In this assay, the binding of 3H-Ro 15-1788 (flumazenil) to the benzodiazepine site is measured. The GABAA-benzodiazepine receptor complex is involved in sedation, epilepsy and convulsions. Out of the 46 extracts tested, seven showed good activity and 10 showed moderate activity. The most active extracts were the ethanolic leaf extracts of Arctopus echinatus, Artemisa afra, four Helichrysum species and Mentha aquatica which all showed good dose-dependent activity.

Alkaloids from Boophane disticha with affinity to the serotonin transporter in rat brain.

Bulbs and leaves of Boophane disticha are used in South African traditional medicine in the treatment of anxiety. Crude extracts of the leaves have shown affinity to the SSRI site on the serotonin transporter in a radioligand binding assay. In this study, two compounds, buphanadrine and buphanamine, were isolated by bioassay-guided fractionation on VLC and preparative TLC. The structures of the compounds were determined by (1)H and (13)C NMR. Fractions were tested for affinity to the serotonin transporter in a binding assay using [(3)H]-citalopram as ligand. The IC(50) values of buphanidrine and buphanamine were 274 microM (K(i)=132 microM) and 1799 microM (K(i)=868 microM), respectively. The two alkaloids were also tested for affinity to the 5HT(1A) receptor, but only showed slight affinity.

Screening of indigenous plants from South Africa for affinity to the serotonin reuptake transport protein.

Seventy five extracts from 34 indigenous plant species used in South African traditional medicine or taxonomically related to these were investigated for their affinity to the serotonin reuptake transport protein, making use of an in vitro serotonin reuptake transport protein binding assay. Aqueous and 70% ethanolic extracts of various plant parts were screened and 45 extracts derived from 15 plant species showed affinity. The affinity of 12 extracts from four plants was characterized as high (more than 50% inhibition at 5, 1, and 0.5 mg/ml). Plant species with high affinity to the serotonin reuptake transport protein included Agapanthus campanulatus, Boophane disticha, Datura ferox and Xysmalobium undulatum. Agapanthus campanulatus yielded high activity in aqueous extracts from leaves and flowers. Boophane disticha showed high activity both in aqueous and ethanolic extracts of leaves and bulbs. Datura ferox showed high activity in aqueous extracts from the seeds and Xysmalobium undulatum showed high activity in the ethanolic extract of the whole plant.

Screening of plants used in southern Africa for epilepsy and convulsions in the GABAA-benzodiazepine receptor assay.

A number of plants are traditionally used to treat mental diseases in South Africa. Aqueous and ethanol extracts of 43 plants that are traditionally used to treat against epilepsy and convulsions, were tested in the GABAA-benzodiazepine receptor binding assay, where the binding of 3H-Ro 15-1788 (flumazenil) to the benzodiazepine site is measured. The GABAA-benzodiazepine receptor complex is involved in epilepsy and convulsions. Out of the 118 extracts tested, one aqueous and 18 ethanol extracts showed activity. The most active extracts were the ethanolic leaf extracts of Rhus tridentata, Rhus rehmanniana and Hoslundia opposita and the ethanolic corm extract of Hypoxis colchicifolia, which all showed good dose-dependent activity.

Acetylcholinesterase enzyme inhibitory effects of amaryllidaceae alkaloids.

Twenty-three Amaryllidaceae alkaloids having several different ring types were evaluated for their acetylcholinesterase enzyme (AChE) inhibitory activity. The alkaloid 1- O-acetyllycorine (IC50 : 0.96 +/- 0.04) showed significant AChE inhibitory activity. In addition, crinine (IC50 : 461 +/- 14), crinamidine (IC50 : 300 +/- 27), epivittatine (IC50 : 239 +/- 9), 6-hydroxycrinamine (IC50 : 490 +/- 7), N-desmethyl-8alpha-ethoxypretazettine (IC50 : 234 +/- 13) N-desmethyl-8beta-ethoxypretazettine (IC50 : 419 +/- 8), lycorine (IC50 : 213 +/- 1), and 1,2-di- O-acetyllycorine (IC50 : 211 +/- 10) had weak activity. Lycorine-type alkaloids were the most active alkaloids with 1- O-acetyllycorine exhibiting inhibitory effects two-fold more potent than that of galanthamine.