Chirumbolo, Salvatore

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CHIRUMBOLO, Salvatore

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Gelsemine and Gelsemium sempervirens L. Extracts in Animal Behavioral Test: Comments and Related Biases

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Salvatore Chirumbolo

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possibility for a reduction of side effects 
due to Gelsemium noxious compounds 
if more selective compounds can be 
identified.
Centesimal (C) dilutions of G. semper-
virens (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 dif-
ferent current tests in one single appara-
tus, in such a way that the emotional status 
of an animal becomes assessable through 
a series of distinct tasks, should contrib-
ute 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 con-
tribution of biases may overwhelm any reli-
able output (Bellavite et al., 2009). The main 
active substance in G. sempervirens causing 
an anxiolytic action is believed to be gelsem-
ine (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) neuros-
teroid synthesis (Venard et al., 2008). The 
stimulatory effects of glycine and gelsem-
ine on 3α,5α-THP production were addi-
tive 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 neuroster-
oids antagonize certain acute effects of 
benzodiazepines and may enhance arousal 
via antagonist or inverse agonist actions 
at the benzodiazepine/GABA(A) recep-
tor complex (Venard et al., 2008). As sev-
eral 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 ani-
mals, 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, how-
ever, 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 labora-
tory 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 fol-
lowed (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 con-
cerning the opportunity to use these mod-
els 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 test-
ing with laboratory animals: actually, none 
of the recently published papers about the 
Gelsemine and Gelsemium sempervirens L. extracts in 
animal behavioral test: comments and related biases
Salvatore Chirumbolo*
Department of Pathology and Diagnostics, University of Verona, Verona, Italy
*Correspondence: salvatore.chirumbolo@univr.it
A commentary on
Dose-effect study of Gelsemium semper-
virens in high dilutions on anxiety-related 
responses in mice
by Magnani, P., Conforti, A., Zanolin, E., 
Marzotto, M., and Bellavite, P. (2010). 
Psychopharmacology (Berl.) 210, 533–545.
Recently reported papers have shown the 
role of the alkaloid gelsemine in behav-
ioral 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 com-
plexity of the Gelsemium toxins involved 
(Lai and Chan, 2009). Gelsemium extracts 
are, in fact, potentially genotoxic. Evidence 
was reported showing that the 95% etha-
nol 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 isola-
tion and identification of several steroids, 
in addition to scopoletin and its deriva-
tives, gelsemine, sempervirine, uvaol, 
and 2-(4-hydroxyphenyl)-ethyl hepta-
decanoate (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 investi-
gated 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 
www.frontiersin.org May 2011 | Volume 2 | Article 31 | 1
General Commentary
published: 16 May 2011
doi: 10.3389/fneur.2011.00031
Greenland, S. (1996). Basic methods for sensitivity 
 analysis of biases. Int. J. Epidemiol. 25, 1107–1116.
Jennions, M. D., and Møller, A. P. (2003). A survey of the 
statistical power of research in behavioural ecology 
and animal behavior. Behav. Ecol. 14, 438–445.
Lai, C. K., and Chan, Y. W. (2009). Confirmation of 
Gelsemium poisoning by targeted analysis of toxic 
Gelsemium alkaloids in urine. J. Anal. Toxicol. 33, 
56–61.
Lewejohann, L., Reinhard, C., Schrewe, A., Brandewiede, J., 
Haemisch, A., Görtz, N., Schachner, M., and Sachser, N. 
(2006). Environmental bias? Effects of housing condi-
tions, laboratory environment and experimenter on 
behavioral tests. Genes Brain Behav. 5, 64–72.
Magnani, P., Conforti, A., Zanolin, E., Marzotto, M., and 
Bellavite, P. (2010). Dose-effect study of Gelsemium 
sempervirens in high dilutions on anxiety-related 
responses in mice. Psychopharmacology (Berl.) 210, 
533–545.
Maren, S. (2001). Neurobiology of Pavlovian fear condi-
tioning. Annu. Rev. Neurosci. 24, 897–931.
McDannald, M. A. (2010). Contributions of the amygdala 
central nucleus and ventrolateral periaqueductal grey 
to freezing and instrumental suppression in Pavlovian 
fear conditioning. Behav. Brain Res. 211, 111–117.
McMillan, F. D. (1999). The placebo effect in animals. J. 
Am. Vet. Med. Assoc. 215, 992–999.
Nakagawa, S. (2004). A farewell to Bonferroni: the prob-
lems of low statistical power and publication bias. 
Behav. Ecol. 15, 1044–1045.
Ramos, A. (2008). Animals models of anxiety: do I need 
multiple tests? Trends Pharmacol. Sci. 29, 493–498.
Schun, Y., and Cordell, G. A. (1987). Cytotoxic steroids 
of Gelsemium sempervirens. J. Nat. Prod. 50, 195–198.
Venard, C., Boujedaini, N., Belon, P., Mensah-Nyagan, 
A. G., and Patte-Mensah, C. (2008). Regulation of 
neurosteroid allopregnanolone biosynthesis in the 
rat spinal cord by glycine and the alkaloidal analogs 
strychnine and gelsemine. Neuroscience 153, 154–181.
Venard, C., Boujedaini, N., Mensah-Nyagan, A. G., and 
Patte-Mensah, C. (2009). Comparative analysis of 
gelsemine and Gelsemium sempervirens activity on 
neurosteroid allopregnanolone formation in the spi-
nal cord and limbic system. Evid. Based Complement. 
Alternat. Med. 5, 80–89.
Wald, C., and Wu, C. (2010). Of mice and women: the bias 
in animal models. Science 327, 1571–1572.
Zhang, Z., Wang, P., Yuan, W., and Li, S. (2008). Steroids, 
alkaloids and coumarins from Gelsemium sempervi-
rens. Planta Med. 74, 1818–1822.
Received: 28 February 2011; accepted: 05 May 2011; pub-
lished online: 16 May 2011.
Citation: Chirumbolo S (2011) Gelsemine and Gelsemium 
sempervirens L. extracts in animal behavioral test: com-
ments and related biases. Front. Neur. 2:31. doi: 10.3389/
fneur.2011.00031
This article was submitted to Frontiers in Neuropharmacol-
ogy, a specialty of Frontiers in Neurology.
Copyright © 2011 Chirumbolo. This is an open-access 
article subject to a non-exclusive license between the 
authors and Frontiers Media SA, which permits use, dis-
tribution and reproduction in other forums, provided the 
original authors and source are credited and other Frontiers 
 conditions are complied with.
 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 phar-
macological and cognitive  interventions 
(Bishop, 2007). Besides randomized blind-
ing procedures, many researchers treat 
animal models with the same experimen-
tal standardization and evaluation applied 
to cells; notwithstanding, animals produce 
effects in response to external stimuli that 
are not so homogenously related to an envi-
ronmental background. Environmental fac-
tors play their significant role. With regard 
to the differences between enriched and 
unstructured housing conditions, overall 
consistent results were achieved by dif-
ferent 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.
RefeRences
Bellavite, P., Magnani, P., Marzotto, M., and Conforti, 
A. (2009). Assays of homeopathic remedies in 
rodent behavioural and psychopathological models. 
Homeopathy 98, 208–227.
Bishop, S. J. (2007). Neurocognitive mechanisms of 
anxiety: an integrative account. Trends Cogn. Sci. 11, 
307–316.
Bonamin, L. V., and Endler, P. C. (2010). Animal models 
for studying homeopathy and high dilutions: concep-
tual critical review. Homeopathy 99, 37–50. [Erratum 
in: Homeopathy 2010, 99, 152].
Chirumbolo, S. (2011). Randomized blinding in 
basic research of homeopathy: some comments. 
Complement. Ther. Med. 19,107–108.
Cohen, J. (1988). Statistical Power Analysis for the 
Behavioural Sciences, 2nd Edn, Hillsdale, NJ: Lawrence 
Erlbaum.
Colloca, L., Petrovic, P., Wager, T. D., Ingvar, M., and 
Benedetti, F. (2010). How the number of learning 
trials affects placebo and nocebo responses. Pain 151, 
430–439.
Dutt, V., Dhar, V. J., and Sharma, A. (2010). Antianxiety 
activity of Gelsemium sempervirens. Pharm. Biol. 48, 
1091–1096.
Enck, P., Benedetti, F., and Schedlowski, M. (2008). New 
insights into the placebo and nocebo responses. 
Neuron 59, 195–206.
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 typi-
cal 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 contrib-
ute 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 rela-
tionships is quite low: the power to detect 
a medium effect is 39–47% and in this case 
only 10–20% of tests exceeded the recom-
mended minimum criterion of 80%, as 
assessed by Cohen and colleagues (Cohen, 
1988; Jennions and Møller, 2003). Biases can 
be unknowingly brought in by research-
ers 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 
Møller, 2003). This is a rational, but worry-
ing, behavior because studies with signifi-
cant 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 preven-
tion of PFC mechanism but also a biases 
analysis should be performed in order to 
assess the reliability of experimental results 
(Greenland, 1996). Recent neuroimag-
ing studies of conditioned fear, attention 
to threat and interpretation of emotion-
ally ambiguous stimuli indicate common 
Frontiers in Neurology | Neuropharmacology  May 2011 | Volume 2 | Article 31 | 2
Chirumbolo Comments on Gelsemium behavioral tests


Salvatore eChirumbolo

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