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A recent discussion expanded the debate about the experimental research on Gelsemium in anxiety. Herbal medicine is widely used in anxiety and mood disorders, often with contradictory evidence, although some authors are yet prompted to promote their full introduction in pharmacology as a promising therapy. Complementary and alternative medicine (CAM) in anxiety is particularly appreciated by individual healthcare, but deserves further investigation, as many critical issues have been recently raised. Comments about the ability of negligible doses of Gelsemium hydroalcoholic extracts to affect gene expression were recently reported.

Plants of the genus Gelsemium have measurable effects on anxiety-like symptoms in laboratory models (Magnani et al., 2010; Liu et al., 2013; Meyer et al., 2013; Jin et al., 2014). Searching for a possible mechanism of action, we found that very low doses and homeopathic dilutions of Gelsemium sempervirens (Gelsemium) modulate the expression of genes involved in neuronal functions (G-protein coupled receptor signaling pathways, calcium homeostasis, inflammatory response and receptors) (Marzotto et al., 2014; Olioso et al., 2014). The commentary's first criticism is that “the search for an involvement of neural genes related to anxiety/depression or mood disorders is biased by the expression of human genes having no orthologs/homologs in mice, where the authors reported evidence about Gelsemium action on behavioral tests in animal anxiety models.” Frankly, we do not see what type of bias can be found in our line of research: Gelsemium is traditionally used as a remedy for anxiety-related symptoms in humans; we have shown that it works in mice models (Magnani et al., 2010; Bellavite et al., 2012), and we wanted to study its mechanism of action at the molecular level, using human neurocyte cell lines. This type of experimental procedure, which addresses various knowledge gaps using both animal studies and in vitro models, is very common in pharmacological studies. Furthermore, the commentary (Chirumbolo, 2014) states that “some genes indicated to be downregulated by Gelsemium 2c, should not be expressed by neuronal cells (e.g., CD163, MPO, C8B, LST1, TREM2, notoriously expressed in immune cell).” Actually, it is well known that neurons express genes of cellular pathways also involved in cytokine/chemokine and immune responses. Genes related to immune and inflammatory responses are expressed in SH-SY5Y cells, as reported by many researchers (Gatta et al., 2011; Toyama et al., 2011; Cui et al., 2012; Xu et al., 2013). Although Gelsemium contains several different compounds (Dutt et al., 2010; Jin et al., 2014), the major active alkaloid of this plant is gelsemine. In the cited commentary we read that “concentration of gelsemine was not assessed, as it was solely calculated on previous spectrometry investigations and new preparations, from ethanol draw extracts, were not further quantified by analytical chemistry.” This statement is misleading because the concentration of gelsemine which we correctly reported (0.021 g/100 ml, corresponding to 6.5 × 10−4 mol/L) (Marzotto et al., 2014, p. 2) was precisely determined by liquid chromatography (not “spectrometry”) in the mother tincture from which the samples were prepared. The concentration of UV-VIS absorbing substances decreased by a factor of 100 at each centesimal dilution step, to become analytically undetectable after a few passages, as we have shown by means of spectrometry in our paper. What we were interested in was to check the accuracy of the procedures as far as possible, not to determine the gelsemine concentration in all subsequent centesimal dilutions. Perhaps we need to remind readers that normally, when one performs a study of dose-response and the concentration in the highest dose and the dilution factor are known, there is no need to determine the concentration of substances in all successive dilutions. Another erroneous criticism which forces us to reply is the statement (Chirumbolo, 2014) that our UV-VIS spectra (Marzotto et al., 2014) “showed a peak at 250 nm caused by contaminating millimolar ethanol in Gelsemium 2c.” This is untrue, and we fail to understand how the writer could have reached such a conclusion, since UV-visible absorption spectra were performed with a double-beam spectrophotometer using drug samples and reference controls, having exactly the same ethanol concentration. Subsequently the commentary claims that “the authors tested a complex mixture of G. sempervirens extract, containing at least about 0.154 mM EtOH at 2c, if dilutions were conducted exactly” and that “concentration of EtOH, set at 30% v/v, faded out to 0.003% in tested dilutions but the authors did not clarify how much for each centesimal dilution in the Methods section.” The writer supposes that high ethanol concentration could have caused “latent apoptosis.” This is confusing and misleading. As clearly indicated in our paper (Marzotto et al., 2014), the final ethanol concentration was 0.03% v/v (p. 2), and “no significant differences in cell viability were observed between cells treated with the ethanol control solution 0.03% (v/v) and untreated cells” (p. 5). Furthermore, as other researchers have found (Do et al., 2013), cell viability of neurocytes is unaffected by doses of ethanol up to 10 mmol/L (0.06%). Low doses of ethanol may influence gene expression, but would have acted in the same way in the Gelsemium and in the control samples. To rule out any misinterpretation of our findings, Figure ​Figure11 gives a brief outline of our microarray experiments. Figure 1 Workflow of the main steps of the DNA-microarray experiments aimed to discover differentially expressed genes in Gelsemium-treated neuronal cells compared with control treated cells. Cultured SHSY5Y cells were incubated for 24 h with the Gelsemium dilutions ... Finally, the commentary (Chirumbolo, 2014) maintains that “Gene array profile of expression following 24 h incubation with Gelsemium 2c, showed down-regulation of 49 genes, namely 87.5% gene array.” It is hard to understand how this 87.5% of genes was calculated, since the 49 genes represent about 0.1% of the genes on the microarray chip. The observation that “many gene products, listed in the expression profile of 56 genes array, such as LOC154872, KIAA0825, LOC150763, C1orf167, have not been identified” is bizarre. The presence of some as yet unidentified sequences in the human genome database is of course well known. We hope that these clarifications will be welcomed in the interests of providing correct and truthful information to readers. In summary, we provided reliable evidence that Gelsemium exerts a prevalently inhibitory effect on a series of neurocyte genes across a wide dose range (Marzotto et al., 2014; Olioso et al., 2014). The effect decreases with decreasing doses, but whole genome expression analysis made it possible to detect statistically significant changes even at extremely low doses and homeopathic dilutions (e.g., 5th and 9th centesimal dilution, corresponding to final gelsemine concentrations of 6.5 × 10−15 mol/L and 6.5 × 10−23 mol/L respectively). More robust conclusions about the role of the genes involved will require determination whether proteins encoded by the affected genes are similarly changed, using proteomic and phosphoproteomic approaches, and/or further studies using purified active plant compounds.

Cisplatin-induced generation of reactive oxygen species leads to acute nephrotoxicity limiting its use in the treatment of various cancers. Gelsemine, an alkaloid isolated from Gelsemium elegans, is known to possess anti-inflammatory and anti-cancer activities. This study was aimed to investigate as to whether gelsemine can serve as a protective agent against cisplatin-induced nephrotoxicity. Male Wistar rats were divided into 6 groups, each with 6 rats. Group 1 served as control and received the vehicles (peanut oil for 14 days and 0.9 % saline on day 14 for gelsemine and cisplatin respectively). Group 2 received a single intraperitoneal injection of cisplatin on day 14. Group 3 and 4 were pretreated with two different doses of gelsemine in addition to cisplatin, and group 5 and 6 received only gelsemine. The effects of gelsemine on cisplatin-induced nephrotoxicity were examined by measuring anti-oxidant enzymes activities, lipid peroxidation, and DNA damage in the kidneys, a well-established model of oxidative damage. Pretreatment of rats with gelsemine caused a significant attenuation of cisplatin-induced DNA and oxidative damages. The blockade of lipid peroxidation and xanthine oxidase activity was accompanied by increased production and/or activity of anti-oxidants, both enzymatic (catalase, glutathione peroxidase, glutathione reductase, and glutathione-S-transferase) and non-enzymatic (GSH). The biomarkers of kidney malfunctioning, creatinine, and blood urea nitrogen were ameliorated. The results of the present study suggest that gelsemine effectively suppressed cisplatin-induced renal injury by improving redox status.

Gelsemium, a small genus of flowering plant from the family Loganiaceae, comprises five species including the popular Gelsemium sempervirens Ait. and Gelsemium elegans Benth., which are indigenous to North America and China/East Asia, respectively. Approximately 120 alkaloids have been isolated and identified from Gelsemium, with the predominant indole alkaloids including gelsemine, koumine, gelsemicine, gelsenicine, gelsedine, sempervirine, koumidine, koumicine and humantenine. Gelsemine is the principal active alkaloid in G. sempervirens Ait., and koumine and gelsemine are the most and second-most dominant alkaloids in G. elegans Benth. Gelsemium extract and its active alkaloids serve a variety of biological functions, including neurobiological, immunosuppressive and antitumor effects, and have traditionally been used to treat pain, neuralgia, anxiety, insomnia, asthma, respiratory ailments and cancers. This review focuses on animal-based studies of Gelsemium as a pain treatment and its mechanism of action. In contrast to morphine, when administered intrathecally and systemically, koumine, gelsemine and gelsenicine have marked antinociception in inflammatory, neuropathic and bone cancer pains without inducing antinociceptive tolerance. Gelsemium and its active alkaloids may produce antinociception by activating the spinal α3 glycine/allopregnanolone pathway. The results of this review support the clinical use of Gelsemium and suggest that its active alkaloids may be developed to treat intractable and other types of pain, preferably after chemical modification. However, Gelsemium is a known toxic plant, and its toxicity limits its appropriate dosage and clinical use. To avoid or decrease the side/toxic effects of Gelsemium, an individual monomer of highly potent alkaloids must be selected, or alkaloids that exhibit greater α3 glycine receptor selectivity may be discovered or modified.

Hydroboration of bicyclo[3.2.1]octa-2,6-diene derivatives occurred exclusively on one olefin, and within this olefin, remarkable regioselective addition of boron to one carbon terminus was observed. This regioselectivity was significantly improved with more electrophilic hydroborating agents. The regioselectivity is attributed to the directive effect of a remote olefin.

Total syntheses of (±)-21-oxogelsemine (3) and (±)-gelsemine (1) are described. Free radical cyclizations play important roles in constructing the tricyclic core (40 → 41) and oxindole (93 → 96) substructures of the target alkaloids, and an isomerization−cyclization strategy (101 → 102) was used to complete construction of the gelsemine cage. Observations made along the way include intramolecular 1,4- and 1,6-hydrogen atom transfers (64 → 65 and 40 → 47) and a free radical cyclization-fragmentation sequence (85 → 86 + 87). A retroaldol−aldol strategy for adjusting oxindole stereochemistry in gelsemine-like structures is also described (72 → 74 and 86 → 87).

The present study examined the antinociceptive effects of gelsemine, the principal alkaloid in Gelsemium sempervirens Ait. A single intrathecal injection of gelsemine produced potent and specific antinociception in formalin-induced tonic pain, bone cancer-induced mechanical allodynia, and spinal nerve ligation-induced painful neuropathy. The antinociception was dose-dependent, with maximal inhibition of 50% to 60% and ED50 values of 0.5 to 0.6 μg. Multiple daily intrathecal injections of gelsemine for 7 days induced no tolerance to antinociception in the rat model of bone cancer pain. Spinal gelsemine was not effective in altering contralateral paw withdrawal thresholds, and had only a slight inhibitory effect on formalin-induced acute nociception. The specific antinociception of gelsemine in chronic pain was blocked dose-dependently by the glycine receptor (GlyR) antagonist strychnine with an apparent ID50 value of 3.8 μg. Gelsemine concentration-dependently displaced H(3)-strychnine binding to the membrane fraction of rat spinal cord homogenates, with a 100% displacement and a Ki of 21.9μM. Gene ablation of the GlyR α3 subunit (α3 GlyR) but not α1 GlyR, by a 7-day intrathecal injection of small interfering RNA (siRNA) targeting α3 GlyR or α1 GlyR, nearly completely prevented gelsemine-induced antinociception in neuropathic pain. Our results demonstrate that gelsemine produces potent and specific antinociception in chronic pain states without induction of apparent tolerance. The results also suggest that gelsemine produces antinociception by activation of spinal α3 glycine receptors, and support the notion that spinal α3 glycine receptors are a potential therapeutic target molecule for the management of chronic pain.

The chemical compound gelsemine is the major active principle of the yellow jasmine (Gelsemium) that is generally claimed as possessing anxiolytic properties based on empirical and indirect knowledge. Surprisingly, gelsemine effect on anxiety has until now received only little attention. Here, we used the well-validated method for anxiety assessment, the elevated plus-maze combined with video-tracking, to measure gelsemine action on rat anxiety-like behavior. Rats were intraperitoneally injected (500μl/daily/7days) with gelsemine (10(-6), 10(-10) or 10(-14)M) or control solution. Diazepam (DZP) was used as positive standard anxiolytic and additional controls were naive rats similarly manipulated except being injected. Gelsemine or diazepam treatment did not affect the number of closed arm entries and rears illustrating the rat general activity. In contrast, gelsemine (10(-6) to 10(-10)M) or DZP increased dose-dependently the number of entries and the percent of time spent in the open arms indicating that gelsemine is an anxiolytic. Consistently, we observed that gelsemine (10(-6) to 10(-10)M) or DZP also decreased dose-dependently the percent of protected stretched attend postures, an ethological index of anxiety-like state. Altogether, our results constitute a solid set of fundamental data directly demonstrating anxiolytic properties of gelsemine. The report also opens new perspectives for the development of safe and effective gelsemine- or Gelsemium-based strategies against pathological anxiety.

Dear Editor, In a recent article, “Dose-effect study of Gelsemium sempervirens in high dilutions on anxiety-related response in mice” (Psychopharmacology 2010, 210: 533–545), Magnani et al. claimed to provide solid evidence that Gelsemium sempervirens centesimal dilutions have anxiolytic-like effects in ICRCD1 mice. This was based on the finding that, with a nonlinear relationship, the centesimal (C) dilution of G. sempervirens 5 C, 9 C and 30 C, but not 4 C and 7 C, modified mice behaviour in the Light Dark test. The result was not confirmed in the Open Field test. We want to raise some doubts about the reproducibility of the results and the reliability of protocols used to evaluate mice behaviour. Reproducibility, the degree of accordance between the results of the same experiment performed independently in the same or different laboratories, is a fundamental prerequisite for acceptance of any experimental evidence. This requirement was not met by the authors, who published another article in 2009, “Homeopathic doses of Gelsemium sempervirens improve the behavior of mice in response to novel environments” (2009a, eCAM, 1-10), in which a highly diluted G. sempervirens solution was evaluated in the same strain of mice (CD1, though purchased from a different producer). G. sempervirens 5 C dilution gave precisely the opposite results to those reported in Psychopharmacology. Diazepam, used as a standard anxiolytic drug, was completely ineffective on mouse behaviour in the Open Field, but increased the time in the light compartment and transitions in the Light Dark test (see Tables 1 and 2, p. 4). When the G. sempervirens 5 C, 7 C and 30 C dilutions were evaluated in another strain of mice (C57BL/6J), again, the opposite effect from that reported by Magnani et al. (2010) was found. G. sempervirens centesimal dilutions modified mice behaviour in the Open Field (Table 3, Bellavite et al. 2009a p. 5). No information was given on how the same centesimal diluted solutions affected C57BL/6J behaviour in the Light Dark test. The fact that the authors did not comment on their previous contradictory findings in Psychopharmacology is a serious omission. Surprisingly, the article by Bellavite et al. was cited just to justify the number of mice per group they were going to use (p. 536, line 17), although how Bellavite et al. computed the minimum sample size they required is absolutely not clear since no sample size by power analyses was done. To be objective, Magnani et al. cited another article by Bellavite et al. (2009b; Homeopathy, p. 534, left column, 10 lines from the bottom). This was a sort of review in which the authors re-proposed the data they had obtained in CD1 mice (omitting those in C57/BL6J mice!) and already published by Bellavite et al. (2009a), together with the data they later submitted to Psychopharmacology. This article was cited as evidence of promising anxiolytic-like activity of G. sempervirens in the Open Field test, without mentioning the failure in the Light Dark test. L. Cervo (*) Laboratory of Experimental Psychopharmacology, Department of Neuroscience, “Mario Negri” Institute for Pharmacological Research, Via La Masa 19, 20156 Milan, Italy e-mail: luigi.cervo@marionegri.it

Centesimal dilutions (5, 9 and 15 cH) ofGelsemium sempervirensare claimed to be capable of exerting anxiolytic and analgesic effects. However, basic results supporting this assertion are rare, and the mechanism of action ofG. sempervirensis completely unknown. To clarify the point, we performed a comparative analysis of the effects of dilutions 5, 9 and 15 cH ofG. sempervirensor gelsemine (the major active principle ofG. sempervirens) on allopregnanolone (3α,5α-THP) production in the rat limbic system (hippocampus and amygdala or H-A) and spinal cord (SC). Indeed, H-A and SC are two pivotal structures controlling, respectively, anxiety and pain that are also modulated by the neurosteroid 3α,5α-THP. At the dilution 5 cH, bothG. sempervirensand gelsemine stimulated [3H]progesterone conversion into [3H]3α,5α-THP by H-A and SC slices, and the stimulatory effect was fully (100%) reproducible in all assays. The dilution 9 cH ofG. sempervirensor gelsemine also stimulated 3α,5α-THP formation in H-A and SC but the reproducibility rate decreased to 75%. At 15 cH ofG. sempervirensor gelsemine, no effect was observed on 3α,5α-THP neosynthesis in H-A and SC slices. The stimulatory action ofG. sempervirensand gelsemine (5 cH) on 3α,5α-THP production was blocked by strychnine, the selective antagonist of glycine receptors. Altogether, these results, which constitute the first basic demonstration of cellular effects ofG. sempervirens, also offer interesting possibilities for the improvement ofG. sempervirens-based therapeutic strategies.

Recently reported papers have shown the role of the alkaloid gelsemine in behavioral science (Dutt et al., 2010). Other issues have yet raised questions about cytotoxicity of Gelsemium compounds: plants belonging to the genus Gelsemium are highly poisonous but toxicological evaluation of suspected poisoning cases has been hampered by the chemical complexity of the Gelsemium toxins involved (Lai and Chan, 2009). Gelsemium extracts are, in fact, potentially genotoxic. Evidence was reported showing that the 95% ethanol extract of Gelsemium sempervirens L. plays an inhibitory activity against human DNA topoisomerase I (Zhang et al., 2008). Phytochemical investigations of this active extract resulted in the isolation and identification of several steroids, in addition to scopoletin and its derivatives, gelsemine, sempervirine, uvaol, and 2-(4-hydroxyphenyl)-ethyl heptadecanoate (Zhang et al., 2008). Actually, cytotoxic steroids of Gelsemium plant were described elsewhere in the past (Schun and Cordell, 1987). G. sempervirens (L.) (Loganiaceae) has been recommended for a long time for the relief of anxiety in traditional folk medicines, although no pharmacological studies evaluated it in this regard, prior to the recently reported evidence of Dutt et al. (2010). The effect of this medicinal plant has been investigated also in complementary, traditional medicine, and ethnopharmacology, with good results in vitro, as very low doses of Gelsemium extracts has shown to reduce anxiety in animal models (Magnani et al., 2010), an evidence that might suggest the possibility for a reduction of side effects due to Gelsemium noxious compounds if more selective compounds can be identified. Centesimal (C) dilutions of G. sempervirens (4C, 5C, 7C, 9C, and 30C) appeared able to change significantly mouse behavioral effects as assessed in the open-field (OF) and light–dark (LD) tests in blind and randomized fashion (Magnani et al., 2010) but effects in the elevated plus-maze, an assay considered to be important in behavioral assessment of potential anxiolytic compounds (Ramos, 2008), have not been assessed. Actually, the physical integration of different current tests in one single apparatus, in such a way that the emotional status of an animal becomes assessable through a series of distinct tasks, should contribute to increased reliability, rapidity, and comprehensiveness in behavioral testing. Although manuscripts in the literature have reported showing an anxiolytic effect of G. sempervirens at concentrations lower than 1.67 ymol/L, criticism may be raised about biases in their experimental context (Venard et al., 2009; Magnani et al., 2010). When a preparation according to the homeopathic pharmacopeia is assayed in a behavioral experimental setting with animals, the contribution of biases may overwhelm any reliable output (Bellavite et al., 2009). The main active substance in G. sempervirens causing an anxiolytic action is believed to be gelsemine (Bellavite, et al.,2009). Gelsemine, is an alkaloid that contains the same functional groups as strychnine and has been shown to increase 3α,5α-tetrahydroprogesterone or allopregnenolone (3α,5α-THP) neurosteroid synthesis (Venard et al., 2008). The stimulatory effects of glycine and gelsemine on 3α,5α-THP production were additive when the two drugs were combined. These results may suggest that glycine and gelsemine, acting via glycine receptor (Gly-R), upregulate 3α,5α-THP biosynthesis in the spinal cord (Venard et al., 2008). Some pilot data have also raised the possibility that chronically administered neurosteroids antagonize certain acute effects of benzodiazepines and may enhance arousal via antagonist or inverse agonist actions at the benzodiazepine/GABA(A) receptor complex (Venard et al., 2008). As several complex issues were raised, the use of Gelsemium in behavioral science should undergo some reappraisal. In this field, a possible main critical topic might be the placebo/nocebo response of animals, able to exert biases on anxiety models. In medicine, the placebo response or placebo effect has often been regarded as a nuisance in basic research and particularly in clinical research, but very few works were related to animal behavior (McMillan, 1999). The latest scientific evidence has suggested, however, that the placebo effect and the nocebo effect, the negative effects of placebo, stem from highly active processes in the brain that are mediated by psychological mechanisms such as expectation and conditioning (Enck et al., 2008). Pavlovian conditioning might justify a placebo effect in housed laboratory animals (McDannald, 2010). Animals which undergo the same stressing procedure for several times (such as intra-peritoneal injections) may relate the physical stressor to other stimuli, such as operator's odor or voice–sound, particularly when the same operator performs the injections. This bias is not always considered, although wash out and blinding randomized protocols are followed (Chirumbolo, 2011). The search for biases in animal behavior research should be a hallmark of any published paper on the topic. Some simple biases, for example animal sex (as researchers routinely use only male animals), have raised criticism concerning the opportunity to use these models to test drugs that would be used by the human female population (Wald and Wu, 2010). Placebo/nocebo response, as an effect of a conditioning/learning mechanism, is almost never considered in behavioral testing with laboratory animals: actually, none of the recently published papers about the anxiolytic activity of G. sempervirens has referred to the possibility of the placebo effect (McMillan, 1999; Lewejohann et al., 2006; Enck et al.,2008; Colloca et al., 2010). Pavlovian fear conditioning (PFC) is a typical behavioral paradigm in which animals learn to predict aversive events: an aversive stimulus (e.g., a sting) is associated with a particular neutral context (e.g., a tone or a smell), resulting in the expression of fear responses to the originally neutral stimulus or context (Maren, 2001). This phenomenon depends on environmental features, so even the room or the time in a day in which PFC is elicited may constitute another bias in behavioral tests (Magnani et al., 2010). The same mechanism could elicit in animals a placebo response. This bias may affect statistical evaluation of outcoming results (Nakagawa, 2004). When plant derivatives are used in behavioral sciences, many factors contribute to make rather tricky the interpretation of the data. For example, intrinsic biases in the statistical computation are often hidden. According to some Authors, the statistical power of behavioral studies to detect relationships is quite low: the power to detect a medium effect is 39–47% and in this case only 10–20% of tests exceeded the recommended minimum criterion of 80%, as assessed by Cohen and colleagues (Cohen, 1988; Jennions and Moller, 2003). Biases can be unknowingly brought in by researchers themselves: they may be disinclined to increase sample sizes when they infer that there is no significant effect to detect and this would also yield a negative correlation between power and p value (Jennions and Moller, 2003). This is a rational, but worrying, behavior because studies with significant results are more likely to be published than those without: this is particularly true in high dilution or homeopathic research (Bonamin and Endler, 2010). Hence, in behavioral tests, not only a randomized double blinding approach and a prevention of PFC mechanism but also a biases analysis should be performed in order to assess the reliability of experimental results (Greenland, 1996). Recent neuroimaging studies of conditioned fear, attention to threat and interpretation of emotionally ambiguous stimuli indicate common amygdala-prefrontal circuitry underlying these processes, and suggest that the balance of activity within this circuitry is altered in anxiety, creating a bias toward threat-related responses. This provides a focus for future translational research, and targeted pharmacological and cognitive interventions (Bishop, 2007). Besides randomized blinding procedures, many researchers treat animal models with the same experimental standardization and evaluation applied to cells; notwithstanding, animals produce effects in response to external stimuli that are not so homogenously related to an environmental background. Environmental factors play their significant role. With regard to the differences between enriched and unstructured housing conditions, overall consistent results were achieved by different experimenters in both laboratories; the reliability of behavioral phenotyping is not challenged seriously by experimenter and laboratory environment as long as appropriate standardizations are met and suitable controls are involved (Lewejohann et al., 2006). Certainly, research on the anti-anxiety potential of Gelsemium alkaloids needs further investigative efforts, in order to comprehend the role of this plant in neuropharmacology.

In traditional Materia Medica, Gelsemium sempervirens (G. sempervirens) is described as a remedy for a variety of anxiety-like psychological and behavioral symptoms, however consistent evidence of its efficacy is lacking. In our recent experimental studies (Bellavite et al., 2009; Magnani et al., 2010), groups of CD1 mice were treated with highly diluted solutions of G. sempervirens, or with conventional anxiolytic drugs, or with a placebo consisting of only the solvent (control groups). The results showed that G. sempervirens reduced anxiety and fear in a manner quantitatively comparable to the effects of normal anxiolytic drugs, but without provoking any sedation side-effects (which instead did occur with buspirone in that experimental set-up). These findings are consistent with those obtained by a group at the University of Strasbourg, which found that the same plant compound had a direct effect on the production of allopregnanolone by rat neurons grown in cell culture plates (Venard et al., 2009). Thus it is likely that the mechanism involved is the regulation of production of endogenous neurosteroids through glycinergic receptors. The chief innovation of this line of research lies in the fact that, until now, natural remedies based on G. sempervirens had been used only on humans, but without consistent evidence of efficacy or with controversial results. Since these remedies also appear to work on animal models and on in vitro systems, this indicates that they do not have merely a “placebo” effect, and that this type of pharmacology using ultra low doses – referred also as “nanopharmacology” – merits further investigation and validation. The “commentary” entitled “Gelsemine and G. sempervirens L. extracts in animal behavioral test: comments and related biases” (Chirumbolo, 2011) is explicitly announced as a commentary on a recent paper from our research group (Magnani et al., 2010). The commentary does not make clear which are the contested points, nor the reason for the imputation of “related biases” in the title of the letter. The author maintains that “no pharmacological studies evaluated anti-anxiety activity of Gelsemium prior to the recently reported evidence of Dutt et al. (2010)” In point of fact, our first results on this subject were published in 2009 (Bellavite et al., 2009). In the first part of the text, the author dwells at length upon the toxicity of the plant and of one of its active principles, gelsemine. These facts are well-known in the literature, but have no bearing on the question of the pharmacological effect on animal behavior, which in the research addressed by this commentary (Magnani et al., 2010), and in others cited therein (Venard et al., 2008, 2009), is accomplished through the administration of extremely low doses that are expected to be non-toxic. According to literature, in emergencies due to Gelsemium poisoning, gelsemine was sustained as the main marker of toxicity and was detected in the urine at concentrations ranging from 10 to 120 ng/ml (Lai and Chan, 2009); as a matter of facts, in our studies, the maximum concentration of gelsemine in the drug samples tested (precisely in the fourth centesimal dilution from mother tincture) was 0.0021 ng/ml and we administered 0.3 ml to 30 g weighing mice, with a further dilution of about 100-fold assuming uniform distribution. In any case, since no clinical data about potential adverse effects of G. sempervirens for chronic use in humans are available, the safety issue needs to be better addressed. The commentary correctly reports that our work has demonstrated anxiolytic effects of high dilutions of G. sempervirens using two behavioral tests, the open field (OF) and light–dark (LD) tests, but then notes that “effects in the elevated plus-maze, an assay considered to be important in behavioral assessment of potential anxiolytic compounds, have not been assessed.” The author of the commentary should know that there exist dozens of tests for animal behavior, out of which each protocol employs, for theoretical and practical reasons, whichever ones the experimenter deems best suited for the purposes of the research. OF (Prut and Belzung, 2003) and LD (Bourin and Hascoet, 2003) are two validated animal models, which allow to acquire various behavioral parameters and are widely employed in neuropsychopharmacology for drug screening (Belzung and Griebel, 2001; Bourin et al., 2007). Classic anxiolytics (benzodiazepines) as well as the newer anxiolytic-like compounds (e.g., serotonergic drugs) and natural compounds (Chen et al., 2004) have been tested using these paradigms. Therefore, the five different behavioral variables extracted from OF and LD in our investigation are sufficient and demonstrative enough of the psychopharmacological effect, although in the view of the results of our study it is not unreasonable to envisage the use of additional complementary tests for future studies on this topic. Immediately afterward in the commentary we read that “Actually, the physical integration of different current tests in one single apparatus, should contribute to increased reliability, rapidity, and comprehensiveness in behavioral testing.” This might at first glance seem a good idea; but in reality, every researcher in the field of experimental psychopharmacology knows that each model must be validated and is designed to evaluate different parameters. No-one has ever dreamed of using an apparatus which “physically integrates” OF, LD, and the elevated plus-maze. The crux of the matter is reached when the author maintains that “criticism may be raised about biases in their experimental context,” citing the works of Venard (Venard et al., 2008, 2009) and of our own group (Magnani et al., 2010). The author dwells at length on the effects of the active principle gelsemine, but puts forward no arguments in support of his claim of possible “bias” in the research, so that this assertion is impossible to discuss or controvert. In the central paragraph of the commentary, we find a treatise on the “placebo” and “nocebo” effects (all well-known phenomena in pharmacology), but not applicable to the work in question, which adopted rigorous double blind controls, consisting of the same solvent as the drug but without the active principle, precisely to rule out these types of effects. The net effect of the drug was always calculated as a percentage relative to the behavioral parameter measured in the mice treated with placebo. Indeed in the discussed study appropriate blinding, dummy medication, positive controls, and similar testing environment for all conditions were used. These measures are be sufficient to minimize placebo/nocebo biases. Finally, the author implies – without demonstrating it – that there might be a bias in the statistics, where he maintains that “according to some Authors, the statistical power of behavioral studies to detect relationships is quite low: the power to detect a medium effect is 39–47% and in this case only 10–20% of tests exceeded the recommended minimum criterion of 80%, as assessed by Cohen and colleagues.” This assertion is in general terms highly debatable, and by no means endorsed by researchers in the field of behavioral psychopharmacology. With respect to our work, it is false (most experiments gave positive results and the ANOVA was highly significant) and ungrounded because it is impossible for Chirumbolo to have computed the 10–20% percent of tests from our data which are supplied as population averages. Moreover, in our work we clearly specify that the power of the study needed to achieve statistical certainty was correctly computed before undertaking the trials. We obviously agree with Chirumbolo's conclusion that “Certainly, research on the anti-anxiety potential of Gelsemium alkaloids needs further investigative efforts, in order to comprehend the role of this plant in neuropharmacology,” and await other experimental contributions, however it certainly does not constitute a criticism of our work.

Gelsemine is one of the major alkaloids from Gelsemium elegans Benth., which has been used as an antitumor remedy in clinic. In this paper, metabolism of gelsemine has been investigated in vitro in phenobarbital-treated rat liver microsomes. The metabolites of gelsemine were separated and evaluated using the flash silica gel column, preparative HPLC, using NMR and MS methods. According to the spectral data, two metabolites, M1 and M2, were identified as 4-N-demethylgelsemine and 21-oxogelsemine, respectively. By the MTT method in vitro, the antitumor activities between gelsemine and its metabolites were compared in the HepG2 and HeLa cell lines. Moreover, the main metabolic pathway was further proposed.

From the medicinal plants Gelsemium, various skeletal type of indole alkaloids were isolated. Along the biogenetic speculation of these alkaloids, some of the structurally complex compounds, involving sarpagine-, humantenine-, koumine-, and gelsedine-type, were prepared from ajmaline and the Gardneria alkaloids. In the second half of this article, recent synthetic studies on the sarpagine type alkaloids and on a principal Gelsemium alkaloid, gelsemine, are reviewed.

Treatment of decalone (11) with acetone or dimethoxymethane and an appropriate acid gave tetrahydropyrans (10) (74%) and (13) (59%), respectively. Similar treatment of indole (6) with dimethoxymethane gave indoline (14) (59%). Attempts to effect similar reactions with indole (4) were unsuccessful

The stereocontrolled total synthesis of gelsemine (1) via 21-oxogelsemine (2) is reported. Our total synthesis features a stereoselective condensation of cyclopropyl carboxaldehyde 11 and 4-iodo-oxindole, a facile construction of the bicyclo(3.2.1) intermediate 20 with a complete control of the stereochemistry by means of a novel application of divinylcyclopropane-cycloheptadiene rearrangement, and an unprecedented silver ion-mediated lactam formation between carbamoyl chloride and enecarbamate.

Total syntheses of (±)-21-oxogelsemine (3) and (±)-gelsemine (1) are described. Free radical cyclizations play important roles in constructing the tricyclic core (40 → 41) and oxindole (93 → 96) substructures of the target alkaloids, and an isomerization−cyclization strategy (101 → 102) was used to complete construction of the gelsemine cage. Observations made along the way include intramolecular 1,4- and 1,6-hydrogen atom transfers (64 → 65 and 40 → 47) and a free radical cyclization-fragmentation sequence (85 → 86 + 87). A retroaldol−aldol strategy for adjusting oxindole stereochemistry in gelsemine-like structures is also described (72 → 74 and 86 → 87).

Hydroboration of bicyclo[3.2.1]octa-2,6-diene derivatives occurred exclusively on one olefin, and within this olefin, remarkable regioselective addition of boron to one carbon terminus was observed. This regioselectivity was significantly improved with more electrophilic hydroborating agents. The regioselectivity is attributed to the directive effect of a remote olefin.

Acomplexmolecularreorganization (1→2), a sequential anionic aza-Cope rearrangement and Mannich cyclization, and an unprecedented intramolecular Heck reaction of the tetrasubstituted double bond of a vinylogous carbamate are key steps in a new total synthesis of (±)-gelsemine (3). MOM=methoxymethyl, DBU=1,8-diazabicyclo[5.4.0]undec-7-ene.

The synthesis of (±)-gelsemine has been completed from tetracyclic intermediate 2 via a stereospecific [3,3]-rearrangement followed by a one carbon excision to convert a δ-lactam ( 13 ) to a γ-lactam ( 19 ).

The total synthesis of (±)-gelsemine (1) is described. A defining phase of the effort involved recourse to a strategic oxetane ring (see compound 25). It was constructed anticipating an intramolecular displacement of the carbon (C17)−oxygen (O4) bond (see product 48). A key intermediate in the stereospecific elaboration of the oxetane linkage was enone 22, which was susceptible to two β-face attacks leading to 24 and, thence, 25. Three sigmatropic rearrangements were employed in building the bridgehead (C20) and the spiroanilide (C7) quaternary centers en route to gelsemine.

The review includes 103 references on the genus Gelsemium , and comprises ethnopharmacology, morphology, phytoconstituents, pharmacological reports, clinical studies and toxicology of the prominent species of Gelsemium . Alkaloids and iridoids constitute major classes of phytoconstituents of the genus. Most popular species of the genus are the Asian G. elegans and the two North American related species, G. sempervirens and G. rankinii . Gelsemium species are categorized under medicinal as well as poisonous plants. Amongst various species, G. elegans and G. sempervirens possess medicinal value, and have been traditionally used as nervous system relaxant. These plants have been explored exhaustively for their anticancer activity. In the concluding part, the future scope of Gelsemium species has been emphasized with a view to establish their multifarious biological activities and mode of actions

This study was designed to investigate the putative anxiolytic-like activity of ultra-low doses of Gelsemium sempervirens (G. sempervirens), produced according to the homeopathic pharmacopeia.Five different centesimal (C) dilutions of G. sempervirens (4C, 5C, 7C, 9C and 30C), the drug buspirone (5 mg/kg) and solvent vehicle were delivered intraperitoneally to groups of ICR-CD1 mice over a period of 9 days. The behavioral effects were assessed in the open-field (OF) and light-dark (LD) tests in blind and randomized fashion.Most G. sempervirens dilutions did not affect the total distance traveled in the OF (only the 5C had an almost significant stimulatory effect on this parameter), indicating that the medicine caused no sedation effects or unspecific changes in locomotor activity. In the same test, buspirone induced a slight but statistically significant decrease in locomotion. G. sempervirens showed little stimulatory activity on the time spent and distance traveled in the central zone of the OF, but this effect was not statistically significant. In the LD test, G. sempervirens increased the % time spent in the light compartment, an indicator of anxiolytic-like activity, with a statistically significant effect using the 5C, 9C and 30C dilutions. These effects were comparable to those of buspirone. The number of transitions between the compartments of the LD test markedly increased with G. sempervirens 5C, 9C and 30C dilutions.The overall pattern of results provides evidence that G. sempervirens acts on the emotional reactivity of mice, and that its anxiolytic-like effects are apparent, with a non-linear relationship, even at high dilutions.

Free radical cyclization of vinylogous urethanes 5 and 12 gave spiro oxindoles 6 and 13 + 14 , in 95% and 84% yields, respectively. Cyclization of projected gelsemine intermediate 19 resulted in the formation of oxindole 22 via a cyclization, 1,4-hydrogen atom transfer, reduction sequence.

From Gelsemium elegans , G. sempervirens and G. rankinii , three new oxindole alkaloids. 19-( R )-hydroxydihydrogelsevirine, 19-( R )-acetyldihydrogelsevirine and 19-( R )-hydroxydihydrogelsemine, together with the known alkaloids 19-( S )-hydroxydihydrogelsevirine, gelsevirine and gelsemine were isolated. Their structures were determined by spectral analysis; the C-19 stereochemistry of 19-( R )-hydroxydihydrogelsevirine was established by single crystal X-ray analysis.

A silicon-tethered intramolecular meta photocycloaddition reaction was utilized to prepare a photoadduct, which was subsequently fragmented using m-CPBA to give a model for the skeleton of the alkaloid gelsemine.

Acomplexmolecularreorganization (1→2), a sequential anionic aza-Cope rearrangement and Mannich cyclization, and an unprecedented intramolecular Heck reaction of the tetrasubstituted double bond of a vinylogous carbamate are key steps in a new total synthesis of (±)-gelsemine (3). MOM=methoxymethyl, DBU=1,8-diazabicyclo[5.4.0]undec-7-ene.

The 95 % ethanol extract of Gelsemium sempervirens showed inhibitory activity against human DNA topoisomerase I (Topo I). Phytochemical investigations of this active extract resulted in the isolation and identification of three new steroids ( 1 - 3), together with eight known compounds 12 beta-hydroxy-5 alpha-pregn-16-ene-3,20-dione ( 4), gelsemine ( 5), sempervirine ( 6), scopoletin ( 7), 7- O- beta- D-glucopyranosylscopoletin ( 8), 7- O- beta- D-apiofuranosyl-(1-->6)- beta- D-glucopyranosylscopoletin ( 9), uvaol ( 10), and 2-(4-hydroxyphenyl)ethyl heptadecanoate ( 11). The structures of the new steroids were determined by extensive NMR and HR-ESI-MS analyses as 21-hydroxy-5 alpha-pregn-16-ene-3,20-dione ( 1), 3-oxoandrosta-16-ene-17-carboxylic acid ( 2), and 3-oxoandrosta-4,16-diene-17-carboxylic acid ( 3). This study suggests that sempervirine ( 6) intercalates to DNA and also inhibits Topo I through modulating the enzyme activity with an IC (50) of 54.5 +/- 15.9 muM.

From the medicinal plants Gelsemium, various skeletal type of indole alkaloids were isolated. Along the biogenetic speculation of these alkaloids, some of the structurally complex compounds, involving sarpagine-, humantenine-, koumine-, and gelsedine-type, were prepared from ajmaline and the Gardneria alkaloids. In the second half of this article, recent synthetic studies on the sarpagine type alkaloids and on a principal Gelsemium alkaloid, gelsemine, are reviewed.

A double conjugate addition between the lithium dianion of ethyl 3-nitropropionate and p-benzoquinone dimethyl ketal afforded a mixture of diastereomeric [3.2.1]bicylooctanones. These products were converted into an advanced intermediate previously carried forward to (+/-)-gelsemine by Fukuyama and Liu.

A convenient one-pot procedure to prepare angularly substituted bicyclic and tricyclic molecules with excellent diastereoselectivity in good yield was developed, by Fe(CO)5 promoted cyclization. Three transformations (complexation, isomerization, and cyclization) were realized in a single operation. The product of this reaction may be a precursor for synthesis of the alkaloid gelsemine.

Oxindole alkaloids gelsemine and 21-oxogelsemine in racemic form are synthesised from sorbic acid.