Introduction
Herbal medicine is widely used in anxiety and mood disorders, often with contradictory evidence [
1], although some authors are yet prompted to promote their full introduction in pharmacology as a promising therapy [
2,
3]. Complementary and alternative medicine (CAM) in anxiety is particularly appreciated by individual healthcare [
4], but deserves further investigation, as many critical issues have been recently raised. [
5,
6]. As a matter of fact, a recent discussion expanded the debate about the experimental research on
Gelsemium in anxiety [
7‐
11]. This Commentary tries to elucidate major issues causing this debate by addressing the numerous aspects raised in comments published elsewhere in the literature.
The anxiolytic property of
Gelsemium plant has been extensively reviewed [
11‐
14]. Raw alcoholic extracts from
Gelsemium sempervirens showed the ability to modify the response of mice in behavioural tests and reduce anxiety [
15]. In this research, the anxiolytic property related to
Gelsemium extracts has been quite exclusively associated with the alkaloid gelsemine [
13,
15,
16]; yet,
Gelsemium plants contain many further alkaloids with anxiolytic potential [
12], thus suggesting that the anti-anxiety activity of
Gelsemium sempervirens may come indifferently from gelsemine, koumine, or gelsevirine or a complex mixture of several active alkaloids [
13]. Actually, plants from the genus
Gelsemium are considered a source of potential anxiolytic substances [
12]. This means that experimental neuroscience based on the possible use of
Gelsemium as a CAM therapy for anxiety shows many difficulties in highlighting a single active principle accounting for the presumptive evidence of efficacy observed in in vitro and animal models. The current debate on
Gelsemium and anxiety includes the many issues exemplified in Table
1, where bias, comments, and replies to comments are thoroughly summarized. A comprehensive neuropharmacology of
Gelsemium should take into consideration any aspect coming from issues described within the reported table.
Table 1
Fundamental issues in the research about Gelsemium and anxiety
Active principles | Gelsemine (3-ethenyl-1-methyl-2,3,3a,7,8,8a-hexahydro-1 h,5 h-spiro[3,8,5-(ethane[1, 1, 2]triyl)oxepino[4,5-b]pyrrole-4,3′-indol]-2′(1′h)-one) was the only active principle described in the behavioural research | | |
• BIAS 1. Gelsemium plant extracts contain other alkaloids with anxiolytic activity (e.g. koumine, gelsevirine) |
• BIAS 2. The anxiolytic activity of Gelsemium alcoholic extracts was not further dissected in order to identify one or more coworking active principles; • BIAS 3. Gelsemine was supposed to be the only active principle working in Gelsemium extracts on the simple basis of previous in vitro pharmacological evidence • BIAS 4. Adverse effects not evaluated |
Solvent and test samples |
Gelsemium extracts were used as a 30 % EtOH/water mixture and further diluted with EtOH and water to have test samples | | |
• BIAS 5. Refs [ 15, 16, 20, 21] started from a raw EtOH/H2O Gelsemium extract containing 30 % alcohol (~50 mM EtOH), then a 1:100 dilution into water with 30 % EtOH and a 1.100 dilution into water (1:10,000) followed. Final dilution contained ~500 μM EtOH, still biologically active |
• BIAS 6. Ethanol is effective on gene expression at molar concentrations as low as 250 μM |
Experimental setting (a): animal model | Animal models: mouse • BIAS 7. Behavioural tests were performed on mice and in vitro confirmatory cellular tests on humans • BIAS 8. Behavioural tests performed did not include specific tests on anxiety, depression, sedation • BIAS 9. Operators treating animals performed behavioural tests | | |
Experimental setting (b): in vitro cell model | In vitro cell model: human neuroblastoma cell line • BIAS 10. Criticism in gene expression performing and interpretation | | |
Pharmacological interpretation | Associated exclusively with gelsemine and considering the allopregnenolone/GABAR pathway • BIAS 11. The anxiolytic activity of Gelsemium may derive from other alkaloids besides gelsemine • BIAS 12. Gelsemine can be anxiolytic through a GlyR/GlyT1-mediated mechanism | | |
Dilutions and ponderal chemistry. Solvents • BIAS 13. Pharmacological interpretation may be hindered by diluted test solutions with negligible amounts of active principles • BIAS 14. Ethanol amount is much more higher than any Gelsemium derived active principles in tested solutions besides Gelsemium 2CH |
Statistics | Statistics with pooling data • BIAS 15. Pooling data projected to retrieve positive evaluation of the mechanism • BIAS 16. Blinded confounders with the same operator in treating and performing test with animals | | |
Most of articles dealing with
Gelsemium in anxiety pertain to CAM therapy. A Pubmed/Medline search of the MESH term
Gelsemium allowed us to retrieve 121 papers from 1945 to date, of which 83 dealt with
Gelsemium in herbal medicine and CAM. The excellent journal Psychopharmacology published at least two papers about
Gelsemium in homeopathy [
15,
17], showing either a cataleptogenic or anxiolytic action by
Gelsemium 30cH, i.e. a theoretical gelsemine concentration less than 6 × 10
−60 mol/L [
15]. In this circumstance, it should be quite difficult to associate any neurologic effect whatsoever with any active molecule present in serially diluted extracts from the
Gelsemium plant. Moreover, comments were raised about the presence of ponderable, significant moles of ethanol added as a co-solvent with water [
9,
10,
18,
19]. While a
Gelsemium 30CH might have negligible traces of possible active principles, its ethanol content would be within the range 0.5–1.0 mM [
18], an occurrence that raised comments about the active molecule in the observed and reported effects [
15,
18‐
21]. These issues prompted this author to address the debate about
Gelsemium in the following step-points.
This article is based on previously conducted studies, and does not involve any new studies of human or animal subjects performed by the author.
Active Principles, Solvent, and Mechanism of Action
Alcoholic raw extracts from plants contain alkaloids and other molecules that may interfere with a plain interpretation of the pharmacology of active principles, due to the complex interaction, either synergistic or competitive, existing between different substances in the raw mixture [
11]. Particularly, gelsemine has been recently associated with a well-defined neuro-pharmacological mechanism related with anxiety. It modulates anxiety in laboratory animals at a sub-micromolar dose range, and in fact, gelsemine doses from 10
−6 to 10
−10 M induce an anxiolytic action in rats in the elevated plus-maze test [
13]. Gelsemine is a
Gelsemium derived alkaloid sharing a chemical and functional kinship with strychnine [
22]. In rat spinal cords, gelsemine showed an additive effect with glycine in increasing the production of the neurosteroid allopregnenolone (3α,5α-tetraidroprogesterone or 3α-idrossi-5α-pregnan-20-one, 3α,5α-THP), which in turn should increase anxiety, due to an increased hippocampal expression of α4βδ GABAA receptors [
23,
24]. 3α,5α-THP is a positive modulator of GABAA receptors and may cause anxiogenic and adverse mood effects in particular circumstances involving steroid withdrawal [
25]. The effect of 3α,5α-THP on GABAA receptors is particularly complex in neuroscience and depends on the many factors related to chronic stress, the expression level of the GABA receptor α4 subunit, the direction of chloride-mediated ionic fluxes created by these target receptors, leading also to a downregulation or dampening in the benzodiazepine ability to modulate this mechanism [
8,
25]. This should suggest that, at least in animal models, the anxiolytic action attributed to gelsemine may be actually caused by other mechanisms, and more caution is requested about a presumptive 3α,5α-THP/GABA relationship with anxiolytic effects. Interestingly, recent reports on the effect of hydroalcoholic extracts from
Gelsemium sempervirens on mouse behaviour showed a marked insensitivity of mice to diazepam [
26]. In this circumstance, criticism was raised about setting and evaluation of mice stress response in behavioural tests [
8,
9]. Furthermore, other alkaloids contained in
Gelsemium plants, such as koumine, have been associated with a 3α,5α-THP/GABA receptor signaling [
26].
Yet, the anxiolytic activity exerted by
Gelsemium might be caused by many further mechanisms. Many
Gelsemium-derived alkaloids, such as kuomine and gelsenicine [
26,
27] exert a nociceptive effect. Particularly, gelsemine acts on chronic pain through the activation of spinal α3 glycin receptors (GlyR) [
22]. This should suggest that the anxiolytic activity associated with
Gelsemium may not directly come from GlyR activation, but most probably from the contribution of activated GlyR on the anziolytic activity of glycine transporter inhibitors [
28]. Therefore, it is very difficult to highlight the neurological mechanism by which
Gelsemium exerts its anxiolytic activity, when
Gelsemium extract is used within a micromolar-millimolar range. Furthermore,
Gelsemium contains a lot of molecules with sedative, anti-depressant activity [
8], for which it is very difficult to ascertain an anxiolytic activity only by widely used, not properly suited behavioural tests [
8,
9,
15]. In this perspective, other components contained in
Gelsemium-derived test solutions such as ethanol, may be significantly involved [
8]. Describing a comprehensible overview of the anxiolytic activity of
Gelsemium extracts, is hampered also by the recent observation that flavonoids, which are present in the
Gelsemium plant, may exert an anxiolytic action [
29]. Furthermore, the involvement of the GABAergic system in anxiety models is yet controversial, because anxiogenic/anxiolytic activity on GABAergic systems may be modulated by different types of orexins [
30]. This strongly suggests that the interpretation of
Gelsemium anxiolytic activity by involving a single, defined mechanism [
15] may be reductive.
A recent behavioural research on ICR-CD1 mice used an hydroalcoholic extract of
Gelsemium sempervirens, which was serially diluted to reach negligible concentrations of potentially bio-active molecules [
15]. ICR-CD1 mice are not particularly suited for behavioural tests compared to the more considered C57BL6 J mouse [
31]. A large number of the laboratory mice sold and used by investigators around the world are considered to be outbred or random-bred. Popular stocks of such mice in the US include CD-1 (Charles River Breeding Laboratories), Swiss Webster (Taconic Farms), and ICR, and NIH Swiss (both from Harlan Sprague–Dawley). Outbred mice are used for the same reasons as F
1 hybrids—they exhibit hybrid vigor with long life spans, high disease resistance, early fertility, large and frequent litters, low neonatal mortality, rapid growth, and large size. However, unlike F
1 hybrids, outbred mice are genetically undefined. Nevertheless, outbred mice are bought and used in large numbers simply because they are less expensive than any of the genetically defined strains. These animals are widely used for behavioural tests. Behavioural tests most commonly used, such as the light dark box test (LDBT) or open field test (OFT), should evaluate time spent at light, without hiding into a small pitch dark hole or walking on the centre of an empty arena, as a measure of stress lacking or anxiety absence for tested animals [
15], yet these tests are used also to evaluate sedation, fear-related stress and depression [
9] and are much less specifically used for anxiety research than others [
8].
Test solutions of
Gelsemium alcoholic extracts were made by diluting 1:100 solutions starting from a raw material containing 30 % or about 50 mM ethanol [
15,
20,
21]. Concentration of gelsemine, a major component of
Gelsemium extract, was calculated as low as 6.5 × 10
−4 M in the fresh hydroalcoholic raw extract, then diluted 1:100 (6.5 × 10
−6 M) in 30 % ethanol (49.93 mmol/L) and significant evidence reported for tested solutions containing an estimated concentration of 6.5 × 10
−20 M gelsemine and 4.99 × 10
−4 M ethanol [
15,
20,
21]. While the final concentration of ethanol (EtOH) at the so-called
Gelsemium 2CH should be as low as 5 × 10
−6 M and gelsemine calculated as 6.5 × 10
−8 M as 2CH means a final dilution 1:10,000, the authors made 1CH (1:100) in 30 % ethanol (50.5 mM EtOH) and 2CH into water (0.505 mM EtOH, i.e. 5.05 × 10
−4 M EtOH) [
15,
20,
21]. Therefore, in
Gelsemium 2CH, the ratio EtOH/gelsemine was about 10,000:1 [
15,
20]. Because any further dilution was made with this approach, this ratio was particularly higher for EtOH with respect to
Gelsemium at 9CH and even more at 30CH. Comments raised about this alkaloid/ethanol disproportion, which suggested a preponderant role from ethanol respect to
Gelsemium components in modifying mice behaviour in a LDBT and OFT [
15], also highlighted why the evidence was scarcely reproducible [
9,
32]. The authors claimed their results as promising and explained
Gelsemium ability to reduce anxiety in mice by an anxiolytic effect attributed to gelsemine and 3α,5α-THP [
15]. In their paper, the minimal effective concentration of gelsemine, estimated by the iterative dilution process from 6.5 × 10
−4 M, was much lower the concentration reported in recent studies [
13,
15].
The same research group recently showed that diluted hydroalcoholic extracts of
Gelsemium were able to affect gene transcription in human neuroblastoma models [
19‐
21]. They reported the same gelsemine concentration previously shown [
20] and a slight reduction in a microarray gene expression model on human SH-SY5Y neuroblastoma cell line with an estimated concentration of gelsemine as low as 6.5 × 10
−9 M, hence within ranges previously reported for rats [
13,
21]. A cognate paper, published in a niche journal in CAM research, confirmed the effect of this gelsemine dosage, but highlighted also a significant effect, though slight, with doses decisively much lower than 6 nM gelsemine [
20]. In both papers, a diluted hydroalcoholic extract of
Gelsemium downregulated the expression of 49/56 [
21] or 45/55 [
20] genes in SH-SYS5 neuroblastoma. Published comments addressed the issue that the effect observed on gene expression might be brought up by EtOH carry-over in the test solution, due to the predominant presence of EtOH respect to any molecule of the starting
Gelsemium extract [
18]. No gene particularly involved in the neurological mechanism underlying the molecular action of
Gelsemium alkaloids was up- or downregulated in the experimental research [
18,
20,
21].