Antinociceptive, Antidepressant, Anxiolytic And Toxicity Studies On Piper Laetispicum C. DC.

Piper laetispicum C. DC. (Piperaceae), popularly known in folk as Xiao Chang-feng, Shan Hu-jiao, Ye Hu-jiao, is an endemic climbing, glabrous plant available in the southern part of China. As a folk medicine, this plant enjoys vast uses for invigorating circulation and reducing stasis, detumescence and analgesic. Besides, the aerial part of P. laetispicum (Hei Shagan in Dai dialect) is widely used in Dai Nationality, one of the 55 Ethnic Minorities of China, to treat epigastralgia, abdominal pain. After scanning more than 100 medicinal plants in our group, we focus on P. laetispicum because of its potential antidepressant activity. Based on this information and the folk use of P. laetispicum, the present study was designed to evaluate the antinociceptive, antidepressant and anxiolytic effects, as well as the mechanism of actions. The toxicity was studied preliminary.Part 1The classical animal models were used in the research of pharmacological activities and mechanism of actions.The acetic acid-induced writhing test, formalin test, hot plate test, immersion test and xylene-induced ear edema test were used to verify the putative antinociceptive activity of P. laetispicum. The essential oils from different organs, the hydroethanol extract, the fractions and 2 individual compounds - laetispicine and d-sesamin were tested in these animal models. The opioid receptor antagonist naloxone hydrochloride was used to test if the opioid receptors are involved in the antinociceptive activity of P. laetispicum. Results:1. The essential oils from stems, leaves, fruits and roots have antinociceptive effects. There exists a quantity-activity relationship between the quantity of sesqui-terpenes and the antinociceptive activity.2. 95% hydroethanol extract promotes a dose-dependent antinociceptive effect, with a mechanism of action which probably involves the participation of both the peripharel and central system. But the peripheral antinociceptive effect is much better than the cemtral antinociceptive activity.3. Among the different fractions, petroleum ether fraction and chloroform fraction are observed significant antinociceptive in acetic acid writhing test. When it comes to the anti-inflammatory activity, all the petroleum ether fraction, chloroform fraction and ethyl acetate fraction have good activity.4. For individual compounds, both laetispicine and d-sesamin have analgesic activity. Laetispicine has a dose-dependent central antinociceptive activity without the involvement of the opioid receptor, but d-sesamin acts mainly peripherally.Conclusions:1. The essential oils have good antinociceptive effects, the sesqui-terpenes compounds are the main compounds responsible for the activity. While the extract free from the essential oil, with amids alkaloids and ligants as the main compounds, also show a significant antinociceptive activity. There might exist a synergic analgesic effect between the sesqui-terpenes and amides and/or ligants.2. Both the total extract and fractions show strong peripheral analgesic activity.3. Leatispicine and d-sesamin are the two main compounds from the organic fractions of P. laetispicum extract. Laetispicine has a dose-dependent central antinociceptive activity without the involvement of the opioid receptor, but d-sesamin acts mainly peripherally.About the antidepressant effect, the study was undertaken to evaluate the influence of hydroethanol extract, the fractions, laetispicine and the derivatives of laetispicine on the duration of immobility in the forced swimming test (FST) and in the tail suspension test (TST). The mechanism underlying the antidepressant action of laetispicine was preliminary studied.Results:1. HEP1 at doses of 120, 240 and 480 mg/kg appears to produce a specific antidepressant-like behavioral effect after acute and chronic treatment.2. The PEF, CHLF and EAF at the given does have a good antidepressant activity in a dose-dependent manner. The antidepressant activity of PEF and CHLF is stronger than EAF. The serotonergic mechanism may not be involved in the antidepressant-like effect of PEF and EAF. However, serotonergic system takes part in, at least partially, the mechanism of action of CHLF in anti-immobility time activity.3. Laetispicine given orally is effective in producing significant dose-dependent antidepressant-like effects, when assessed in FST and in TST. Our data demonstrate that the activation of the opioid system and serotonergic mechanism seem unlikely to be involved in the antidepressant-like effect of laetispicine, while the L-arginine-nitric oxide pathway might be partially involved in the antidepressant effect of laetispicine.4. A series of laetispicine derivatives were synthesized and evaluated as potential antidepressants. After acute administration, ysy-5, ysy-7, ysy-10, ysy-11, ysy-12, ysy-16 show significant activity. After 7 days administration, ysy-5, ysy-7, ysy-10 and ysy-16 have better antidepressant activity without any drug tolerance.Conclusions:1. HEP1 produces a specific antidepressant-like behavioral effect without drug tolerance after sub-chronic administration.2. Serotonergic system takes part in the mechanism of action of CHLF in anti-immobility time activity, but not PEF and EAF.3. The opioid system and serotonergic mechanism seem unlikely to be involved in the antidepressant-like effect of laetispicine, while the L-arginine-nitric oxide pathway might be partially involved in the antidepressant effect of laetispicine. 4. Among the derivatives, ysy-7, ysy-10 and ysy-16 have the potential for further development. And these data give us some information about the structure and activity relationship.5. The mechanism of actions of extract, fractions, laetispicine and derivatives deserve further research.A series of experiments are designed to verify the anxiolytic effect of ethyl acetate extract and laetispicine in the light/dark test, elevated plus maze test and hole-board test. In addition, the mechanism of action of the extract and laetispicine were preliminary studied using flumazenil, an antagonist of BZD receptor.Results:1. EAF and laetispicine have anciolytic activities.2. EAF at 240 mg/kg has an obvious anxiolytic activity.3. At the given doses, laetispicine at 10 mg/kg has a anxiolytic activity, while no significant activity was observed in laetispicine at the doses of 5 and 20 mg/kg in three animal models.4. Flumazenil didn't block the anxiolytic effect of EAF and laetispicin.Conclusions:1. The anxiolytic activity of AEF is weak.2. The dose response curve of laetispicine in the three animal models is bell-shaped, which maybe has a relationship with its mechanism of action.3. BZD receptor isn't involved in the mechanism of actions of EAF and laetispicine.Open field test was arranged to avoid the effects on locomotor activity caused by central nervous system stimulants. The results showed that all the active doses in these three pharmacological researches did not significantly change locomotor activity. Part 2The acute and chronic oral toxicity effects of EAF extract of P. laetispicum stems was designed in mice to get some basic toxicity information.Results:1. The values of LD50 were 1530.0 and 538.0 mg/kg for oral and intraperitoneal administration of the EAF extract, respectively.2. After 90 days treatment, there are no significant differences in body weight gain or behavior in mice between the drug-treated groups and the vehicle group, while there is a significant increase in liver weight in 500 mg/kg group.3. After 90 days administration, the locomotor and memory abilities are not affected.Conclusions:1. Toxicity in EAF is weak.2. Liver might be the target organ in the chronic administration.3. Chronic administration doesn't affect the locomotor activity and memory ability.In the present research work, 12 different animal models are used to verify the antinociceptive, antidepressant and anxiolytic effects of Piper laetispicum C.DC., an endemic medicinal plant in southern China, as well as the preliminary toxicity research. The data obtained will be the basic scientific information for the further research and development of the fractions extracts, laetispicine and derivatives of laetispicine.