My opinion

By Dr. Salvatore Chirumbolo
Corresponding Author Dr. Salvatore Chirumbolo
Department of Medicine, University Laboratory of Medical Research-Policlinico GB Rossi, piazzale AL Scuro 10 - Italy 37134
Submitting Author Dr. Salvatore Chirumbolo

Gelsemium, Mouse behavior, Neuroscience, Behavioral test, Alcohol, Anxiety, Bias

Chirumbolo S. Behavioral Research with Homoeopathic Remedies from Plant Extracts: Biases and Comments. WebmedCentral COMPLEMENTARY MEDICINE 2011;2(10):WMC002349
doi: 10.9754/journal.wmc.2011.002349
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Submitted on: 20 Oct 2011 10:01:05 AM GMT
Published on: 21 Oct 2011 03:43:12 PM GMT

My opinion

Complementary and alternative medicine (CAMs) in behavioural science has raised several comments, due to the presence of a placebo/nocebo response (Davidson et al, 2011; Chirumbolo, 2011; Bellavite, 2011; Paris A, 2011 ); this issue represents a puzzling question for behavioural science, because placebo effects, in randomized placebo-controlled clinical trials (RCTs) on classical homeopathy, have shown not to be larger than placebo effects in conventional medicine (Nuhn et al, 2010). In this perspective, placebo response appears rather significant in a behavioural or psychiatric clinical outcome and prompts to suggest an obvious question: do commercial homeopathic remedies act simply by a placebo mechanism or not? Researchers tried to overcome this snag by considering animal models. However, a placebo/nocebo response has been shown also for laboratory animals (McMillian, 1999; Enck et al, 2008) and biases in experimental settings are always in ambush. Actually, a further exploration about the nature of the placebo responses in clinical trials has to be made and, moreover, major questions for future research such as the relationship between expectations and conditioning in placebo effects, which have been shown in animals, the existence of a consistent brain network for all placebo effects, the role of gender in placebo effects, and the impact of getting drug-like effects without drugs, have to be addressed (Enck et al, 2008)
Biases in this context may arise. The use of very diluted solutions of plant extracts, such as Gelsemium sempervirens, an approach which was called erroneously as nanopharmacology (Bellavite, 2011; Seifulla, 2010), includes ethanol in the experimental setting. A recently reported commentary maintained that the anxiolytic effect of diluted doses of Gelsemium sempervirens did not provoke any sedation side-effect (Bellavite, 2011). However, as each dose used in his experimental setting, contained 0.3% ethanol and as each mouse underwent i.p. injections for 9 days, a simple calculation can be made: each subject received throughout the whole period a total amount of 7.10 mg ethanol, considering an administration of 1.0 ?l/injection and ethanol density = 0.789 g/cm3 (Magnani et al, 2010; Bellavite et al, 2009). Although some reports has shown that low doses of ethanol might increase the locomotor activity in mice, recent reported evidence in rats suggested that this dose of alcohol may result in possible sedation, though injected in different times. In rats a dose of 0.25 g/kg EtOH has been shown to be the lowest dose able to decrease significantly locomotion in these rodents (Chuck et al, 2006). Whether considering as 500 g the mean body weight of a male subject, this dose should correspond to 125 mg of alcohol; a male mouse is about 1/25 of a male rat, so a mouse receiving about 5 mg of alcohol, might undergo comparable sedation effects. The experimental setting planned and performed by some authors used ethanol in each tested sample, including controls; this should assure that sedation would be carried throughout the analysis, without influencing the net outcome of the overall experiment. Bias arises because ethanol is a pharmacological compound as like as gelsemine or buspirone (Bellavite et al, 2009; Magnani et al, 2010). Rodents response to ethanol may be highly variable, as like as humans (Fraser et al, 1995), because of the intra-individual variability in detoxifying enzymes and non-linear BK channels function (Nelson et al, 2004; Treistman et al, 2009); its pharmacokinetics is complex, it does not depend on mouse strain and administration route, it has a dose-dependent linear increase in alcohol concentration in the plasma and brain and non linear or parabolic increase in the area under ethanol pharmacokinetic curve in tissues (Golovenko et al, 2001). In the experimental conditions reported elsewhere by others (Bellavite et al, 2009; Magnani et al, 2010), it is quite difficult to assess if ethanol might exert a significant effect on pharmacokinetics of other drugs such as ergot alkaloids contained in Gelsemium plant extracts, but what it is well known is that ethanol possesses a significant influence on plasma pharmacokinetics of any drug, in a general way (Lennernas, 2009). Furthermore, the effects of ethanol are often biphasic (stimulatory/inhibitory) as like as low doses of a plant extract (Calabrese, 2008), so rendering more complex any possible interpretation of the assay (Middaugh et al, 1987; Crabble et al, 1982). The association of ethanol with a different drug might change dramatically the fashion by which the drug operates in the behavioral test and a molecular or cellular comparison with the same molecule diluted into water (Bellavite, 2011, Venard et al, 2009) should not have been made, unless considering the introduction of a set of controls without ethanol.
Furthermore, within this experimental condition open field test (OFT) and light-dark box test (LDT) were unable to discriminate sedation or anxiolysis by evaluating locomotor or exploratory activity by alone; furthermore, the reference control buspirone, showed sedation side effects (Magnani et al, 2010; Bellavite, 2011). On the contrary, Venard and colleagues do not include ethanol by no means in their research and the experimental approach dealt with an in vitro research on mice tissue slices (Venard et al, 2009). Gelsemium sempervirens Ait. alcoholic extract contains many other ergot alkaloids besides gelsemine, most of which exert many depressant and sedative effects (Duke, 1992; Gahlot et al, 2011). Furthermore, when low doses of a plant-derived compound are used, hormetic effects may arise. Hormetic mechanisms have been described by using a broad panel of behavioral tests; the analysis revealed that hormetic-like biphasic dose-response were commonly observed across all screening tests (Calabrese, 2008). These issues normally hamper a clear description of the anxiety-like behavior in tested animals, especially if environmental conditions are considered (Lewejohann et al, 2006). For example, dark box in LDT may be felt by mice as an aversive environment, so forcing the animal in spending more time in the lit arena and the lit area, if has the same light exposure of the whole environment, might do not elicit any aversive stimulus (Magnani et al. 2010; Hascoet and Bourin, 1998; Bourin and Hascoet, 2003). These considerations appear very interesting when anxiety models are addressed with laboratory animals. Which is anxiety in laboratory rodents? Is the term anxiety interchangeable with fear, stress, panic or danger sensitivity? In animals, fear is an adaptive response that has evolved to provide protection from potential harmful environments. and fear-related behaviors in mice have long been investigated as potential models of anxiety disorders (Hettema et al, 2011). When fear is disproportionate in facing the harmful situation, it can lead to an anxiety disorder (Graham et al, 2011). In laboratory animals, such as mice or rats, fear may be acquired when a neutral conditioned stimulus is paired with an aversive unconditioned stimulus and, usually, after several such pairings, the subjects is able to learn that the conditioned stimulus elicits several fear responses: in this circumstance, anxiety may arise (Graham et al, 2011). This possibility occurs when the same operator makes serial injections and performs behavioral tests (Magnani et al, 2010). Several reports dealing with herbal extracts in animal anxiety models are limited to the simplest standardized behavioral tests, which are indicated to measure locomotion and exploratory tendency as main parameters of a non-anxious behavior: in this context a decrease in these two parameters might be associated with other hallmarks, such as sedation or depression, then demonstrating that the complex mixture of compounds contained in the alcoholic plant extract may show many different pharmacological effects. Pavlovian conditioning paradigms have become important model systems for understanding the neuroscience of behavior, especially in rodents. In particular, research about the extinction of Pavlovian fear responses is yielding important information about the neural substrates of anxiety disorders, such as phobias and post-traumatic stress disorder (PTSD), even in humans. An advantage of the fear extinction model is that comparison of animal studies should suggest a considerable similarity between the neural structures which are involved in extinction in rodents and in humans. These studies allow to understand the neural mechanisms underlying behavioral interventions that suppress fear, including exposure therapy in anxiety disorders (Chung et al, 2009). Fear and anxiety appear, therefore, as two different and strictly related paradigms in neuroscience. In laboratory, several behavioral tests are available to ascertain if the researcher is investigating a fear extinction mechanism, an anxiety disorder or an extinction of both, due to a pharmacological treatment. One good test is elevated plus-maze. Fear can be measured as a decreased percentage of time spent on open-arm exploration in the elevated plus-maze and can be potentiated by prior inescapable stressor exposure, although not by escapable stress. In this case, the application of fear-potentiated plus-maze behavior has several advantages as compared to more traditional animal models of anxiety, such as LDT or OFT (Magnani et al, 2010). The traditional, elevated plus-maze is able to measure innate fear of open spaces but a fear-potentiated plus-maze behavior should reflect an enhanced anxiety state, called as allostatic state. This typical "state of anxiety" can be defined as an unpleasant emotional arousal in face of threatening demands or dangers (Korte and De Boer, 2003). Actually, a cognitive appraisal of threat is a prerequisite for the experience of this type of emotion. Another hallmark of this test is that depending on the stressor used this enhanced anxiety state can last from 90 min to 3 weeks. Stress effects are more severe when animals are isolated in comparison to group housing. In such a system drugs can be administered either in the absence of the original stressor or after stressor exposure. As a consequence, retrieval mechanisms are not affected by drug treatment. This fear-potentiated plus-maze behavior is sensitive to proven/putative anxiolytics and anxiogenics which act via mechanisms related to the benzodiazepine-gamma-aminobutyric acid receptor, but it is also sensitive to corticotropin-releasing receptor antagonists and glucocorticoid receptor antagonists and serotonin receptor agonists/antagonists complex (Korte and De Boer, 2003). For those reasons, fear-potentiated plus-maze behavior is very robust, experiments can easily be replicated in other labs and the mechanism can be measured both in male and female individuals. In this strategy, neural mechanisms involved in contextual fear conditioning, fear potentiation and state anxiety can be studied, so rendering fear-potentiated plus-maze behavior a valuable measure in the understanding of neural mechanisms involved in the development of anxiety disorders and in the search for novel anxiolytics (Korte and De Boer, 2003; Korte and De Boer, 1999; Grahn et al, 1995). This assumption would like to suggest to better evaluate different behavioral tests in investigating anxiety-like models in animals, attempting to elicit biases at the lowest frequency possible, not to create a possible “integrate behavioral assay” (Bellavite, 2011). For the many reasons previously indicated, the interpretation of behavioral tests includes many tricky issues, which asks for further investigation (Chirumbolo, 2011). CAMs in psychiatry or behavioral disorders, while accounting on animal models derived evidence, may contain biases due to the high complexity of the addressed issues. Statements and comments about the possible effectiveness of low concentrated alkaloids from alcoholic plant extracts have to be reappraised and evaluated at the light of bias analysis (Podsakoff et al, 2003). Anxiety-like models in laboratory animals such as rodents contain many unresolved and puzzling aspects that merit to be explained by using increasingly sophisticated approaches, aiming at not lapsing into easy conclusions.


1. Bellavite P. Gelsemium sempervirens and animal behavioral models. Front Neurol 2011; 2:56 DOI: 10.3389/fneur.2011.00056
2. Bellavite P, Magnani P, Marzotto M, Conforti A. (2009) Assays of homeopathic remedies in rodent behavioural and psychopathological models. Homeopathy 98:208-227
3. Bourin M, Hascoet M. The mouse light/dark box test Eur J Phrmacol 2003; 463 (1-3):55-65
4. Calabrese EJ. An assessment of anxiolytic drug screening tests: hormetic dose responses predominate. Crit Rev Toxicol. 2008;38(6):489-542.
5. Chuck TL, McLaughlin PJ, Arizzi-LaFrance MN, Salamone JD, Correa M. Comparison between multiple behavioral effects of peripheral ethanol administration in rats: sedation, ataxia, and bradykinesia. Life Sci. 2006 Jun 6;79(2):154-61.
6. Chang CH, Knapska E, Orsini CA, Rabinak CA, Zimmerman JM, Maren S. Fear extinction in rodents. Curr Protoc Neurosci. 2009 Apr;Chapter 8:Unit8.23
7. Chirumbolo S. Gelsemine and Gelsemium sempervirens L. Extracts in Animal Behavioral Test: Comments and Related Biases. Front Neurol. 2011;2:31.
8. Crabble JC jr, Johnson NA, Gray DK, Kosobud A, Young ER. Biphasic effects of ethanol on open-field activity: sensitivity and tolerance in C57BL/6N and DBA/2N mice. J Comp Physiol Psychol 1982; 96(3):440-51
9. Davidson JR, Crawford C, Ives JA, Jonas WB. Homeopathic treatments in psychiatry: a systematic review of randomized placebo-controlled studies. J Clin Psychiatry. 2011 Jun;72(6):795-805.
10. Duke, JA. Handbook of phytochemical constituents of GRAS herbs and other economic plants. 1992, Boca Raton, FL. CRC Press
11. Enck P, Benedetti F, Schedlowski M. New insights into the placebo and nocebo responses. Neuron 2008; 59(2):195-206
12. Fraser AG, Rosalki SB, Gamble GD, Pounder RE. Inter-individual and intra-individual variability of ethanol concentration-time profiles: comparison of ethanol ingestion before or after an evening meal. Br J Clin Pharmacol 1995; 40(4):387-92
13. Gahlot K, Abid M, Sharma A. Pharmacological evaluation of Gelsemium sempervirens roots for CNS depressant activity. Int J Pharmatech Res 2011; 3(2):693-697
14. Golovenko NY, Zhuk MS, Zin’kovskii VG, Zhuk OV, Kopanitsa MV. Pharmacokinetics of ethanol in mice with different alcohol motivation. Bull Exp Biol Med 2001; 132(3):852-5
15. Graham BM, Milad MR. The Study of Fear Extinction: Implications for Anxiety Disorders. Am J Psychiatry. 2011 Aug 24, in press, DOI: 10.1176/appi.ajp.2011.11040557
16. Grahn, R. E., Kalman, B. A., Brennan, F. X., Watkins, L. R., and Maier, S. F. The elevated plus-maze is not sensitive to the effect of stressor controllability in rats. Pharmacol. Biochem. Behav. 1995; 52, 565–570.
17. Hascoet M, Bourin M. A new approach to the light/dark test procedure in mice. Pharmacol Biochem Behav 1998; 60(3):645-53
18. Hettema JM, Webb BT, Guo AY, Zhao Z, Maher BS, Chen X, An SS, Sun C, Aggen SH, Kendler KS, Kuo PH, Otowa T, Flint J, van den Oord EJ. Prioritization and Association Analysis of Murine-Derived Candidate Genes in Anxiety-Spectrum Disorders. Biol Psychiatry. 2011, in press, DOI:10.1016/j.biopsych.2011.07.012
19. Lennernas H. Ethanol-drug absorption interaction: potential for a significant effect on the plasma pharmacokinetics of ethanol vulnerable formulations. Mol Pharmaceut  2009; 6(5):1429-1440
20. Lewejohann L, Reinhard C, Schrewe A, Brandewiede J, Haemisch A, Gortz N, Schrachner M, Sachser N Environmental bias? Effecs of housing conditions, laboratory environment and experimenter on behavioral tests Genes Brain Behav 2006; 5(1):64-72
21. Korte, S. M., De Boer, S. F., and Bohus, B. Fear- potentiation in the elevated plus-maze test depends on stressor controllability and fear conditioning. Stress 1999; 3, 27–40.
22. Korte SM, De Boer SF. A robust animal model of state anxiety: fear-potentiated behaviour in the elevated plus-maze. Eur J Pharmacol. 2003 Feb 28;463(1-3):163-75.
23. McMillian FD. The placebo effect in animals J Am Vet Med Assoc 1999; 215(7):992-9
24. Magnani P, Conforti A, Zanolin E, Marzotto M, Bellavite P. Dose-effect study of Gelsemium sempervirens in high dilutions on anxiety-related responses in mice. Psychopharmacology (Berl). 2010 Jul;210(4):533-45
25. Middaugh, L. D., Boggan, W. O., and Randall, C. L.. Stimulatory effects of ethanol in C57BL/6 mice. Pharmacol. Biochem. Behav. 1987; 3:421–424.
26. Nelson D, Zeldin D, Hoffman S, Maltais S, Wain H, Hester M, Nebert D. Comparison of cytochrome P450 (CYP) genes from the mouse and human genomes, including nomenclature recommendations for genes, pseudogenes and alternative-splice variants Pharmacogen 2004; 14(1): 1-18
27. Nuhn T, Ludtke R, Geraedts M. Placebo effect sizes in homeopathic compared to conventional drugs-a systematic review of randomised controlled trials. Homeopathy 2010;99(1):76.82
28. Paris A, Schmidlin S, Mouret S, Hodaj E, Marijnen P, Boujedaini N, Polosan M, Cracowski JL, Effect of Gelsemium 5CH and 15 CH on anticipatory anxiety: a phase III, single-centre, randomized, placebo-controlled study. Fundam Clin Pharmacol 2011 Sept 28. DOI 10.1111/j.1472-8206.2011.00993.x
29. Podsakoff PM, MacKenzie SB, Lee JY, Podsakoff NP Common method biases in behavioral research: a critical review of the literature and recommended remdies J Appl Psychol 2003; 88(5):879-903
30. Se?fulla RD. [Nanopharmacology: not a constituent part of homeopathy]. Eksp Klin Farmakol. 2010 Jul;73(7):40-1.
31. Treistman SN, Martin GE. BK channels. Mediators and models for alcohol tolerance. Trends Neurosci. 2009; 32(12):629-37
32. Venard C, Boujedaini N, Mensah-Nyagan AG, Patte-Mensah C. Comparative Analysis of Gelsemine and Gelsemium sempervirens Activity on Neurosteroid Allopregnanolone Formation in the Spinal Cord and Limbic System. Evid Based Complement Alternat Med. 2009 Jul 23. Article ID 407617, DOI:10.1093/ecam/nep083.

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