The present study sought to analyze the behavioral effects of CZZX, which is used to treat “nervous diseases” in traditional Chinese medicine [11
]. The results showed that CZZX exhibited anxiolytic-like activity and did not induce sedative side effects. The binding assay suggested that the anxiolytic effects of CZZX may be attributable to the modulation of GABAA
receptors. Altogether, our results lend support to the traditional use of CZZX in Chinese folk medicine. The present findings may also provide important leads for the development of potent and selective anxiolytic agents.
When the animals were treated with the higher doses of CZZX (1.5 and 3
g/kg) for 10 days, anxiety-like behavior in the elevated plus maze was significantly attenuated, although the low dose (0.75
g/kg) had no effects on the open-arm indices, suggesting that CZZX possesses anxiolytic effects in the elevated plus maze paradigm. Behavior in the elevated plus maze is related to the natural aversion that rodents have for elevated and open spaces. Anxiolytic drugs shift the behavioral response toward exploration of the open arms [18
]. The main potential confounding factors in this model are changes in basal locomotor activity, which can be inferred from the total number of entries. CZZX did not alter total arm exploration in the elevated plus maze, suggesting that this drug induces specific anxiolytic-like effects.
To further strengthen these data, we tested the anxiolytic-like effects of these treatments in the VCT, which involves the suppression of punished responses. The behavioral suppression induced by shocks in the VCT is attenuated by anxiolytic drugs [19
]. One drawback of this paradigm, however, is that drugs that induce antinociceptive effects or increase water consumption may yield false-positive results. The doses of 1.5 and 3
g/kg CZZX increased the number of punished licks (i.e., induced anxiolytic-like effects), but none of the doses altered the number of unpunished licks. Thus, this is unlikely to be a confounding factor in the assessment of anxiolytic-like effects. Moreover, CZZX was ineffective in the acute thermal pain test, excluding changes in nociceptive threshold as confounding factors.
The results obtained in the open field test showed that chronic CZZX treatment did not affect the distance traveled or emotional behavior (i.e., rearing, grooming, and defecation), suggesting that this compound may not produce undesirable sedative side effects. These results indicate that repeated treatment with CZZX may significantly improve anxiety-like behavior without producing sedative side effects. Many individual herbal preparations of CZZX and their major constituents, including Valeriana jatamansi
], Ziziphus jujuba
], Albizia julibrissin
], and Medulla junci
], exert anxiolytic or sedative effects in various animal models. Given that the anxiolytic-like effects of CZZX observed in the present study were generated from the effects of individual herbs, one may expect that CZZX has superior effects in ameliorating anxiety compared with individual herbal preparations.
In traditional Chinese prescriptions, herbs are usually mixed before extraction. However, the four herbs contained in the formula are all sensitive to water temperature. Some scientific references and experimental evaluations of the isolation of the active principles of each plant have been provided [25
], and we extracted the four herbs individually using different solvents and then mixed the extracts according to the ratio of crude medicines in the CZZX prescription. Preliminary pharmacodynamic studies are being performed in our laboratory to investigate anxiolytic-like effects in mice. We found that these four herbs that were extracted individually exhibited higher potency in anxiolytic tests than the four herbs mixed before extraction (data not shown). Therefore, these four herbs in the formula were extracted individually in the present study.
GABA is the most important inhibitory neurotransmitter in the human central nervous system. Compelling evidence suggests an imbalance between excitatory and inhibitory neurotransmitters in the pathophysiology of convulsions, anxiety, and sleep [26
]. The GABAA
receptor system is the main fast-acting inhibitory neurotransmitter system in the brain and the pharmacological target for many drugs used clinically to treat anxiety disorders and epilepsy. GABAA
receptors are heteromeric GABA-gated chloride channels. The BZD site on GABAA
receptors modulates the inhibitory effects of GABA [27
]. Benzodiazepine site agonists, such as diazepam, increase the GABA-induced chloride channel opening frequency, exerting anxiolytic, anticonvulsant, muscle relaxant, sedative-hypnotic, and cognition-impairing effects [28
] and rendering these agonists the most important GABAA
receptor-modulating drugs in clinical use. For this reason, discovering plants that enhance GABA affinity for the GABAA
receptor is important.
In the present study, diazepam was used as a positive control. As expected, it increased activity in the open arms of the elevated plus maze and number of punished licks in the VCT, confirming its anxiolytic actions. Many benzodiazepines and related compounds that bind to receptors in the central nervous system have been identified in certain plant extracts [29
]. In the search for the underlying mechanism of the anxiolytic-like effect of CZZX, we performed a complementary assay to investigate whether the effects of CZZX were attributable to its action at GABAA
receptors. The binding studies were conducted using the GABA antagonist [3
H] Ro 15-1788 (flumazenil). In the GABAA
-BZD binding assay, CZZX displaced [3
H] Ro 15-1788 to brain synaptosomal membranes, indicating that CZZX had effective concentration-dependent binding activity and suggesting central BZD-like activity. However, the EC50
for CZZX was approximately 0.5
mg/mL, and further pharmacokinetic and pharmacodynamic studies are needed in the future.
In summary, the present study showed that CZZX elicited strong effects on anxiety-like behavior, likely mediated by its BZD-like activity. The extent of anxiolytic activity was comparable to the effects of diazepam, but CZZX may be devoid of undesirable side effects, such as sedation. Therefore, CZZX is a promising candidate for the treatment of anxiety-like disorders. Studies are being performed in our laboratories to isolate the active principles of CZZX and determine the specific effects of CZZX on the central nervous system and its underlying mechanism of action.