Animals
Seven weeks old male albino rats of Charles Foster strain (100–150 g) were used. The rats, purchased from JNMC Animal House, Aligarh, were housed in groups of eight in standard cages with free access to solid pellet diet (Gold Mohar, Lipton-India, Ltd.) and tap water except during the time of experiment. The "Animal Observation Room" was controlled so that the light dark cycle (light period 6.00 am to 6.00 p.m.; 12 hours) and temp. (23 ± 2°C) were nearly constant. The study was approved by the JNMC Animal Ethics Committee.
Drugs
Morphine sulphate (Sigma Chemicals Co., USA).
Delphinium denudatum Wall. (D. d). The tuberous roots of D.d. in the dried form were procured from Dawakhana Tibbia College, Aligarh Muslim University, Aligarh. These were identified by comparison for macroscopic and microscopic characters with authentic specimens of D. d. kept at the Botanical Survey of India and the Forest Research Institute, Dehradun. Due to the presence of various alkaloids in roots, the extraction was carried out by mixing the powdered roots with 1:3 w/v in absolute alcohol by Soxhlets apparatus for 72 hours. Yield of extract was 7.43 % w/w in terms of dried starting material. It was sticky in character, pilular, brown and of pleasant smell.
Treatment schedule
The rats were divided into 6 groups of 8 animals each. All the animals were treated with 0.9% saline, D.d. extract using distilled water as the dose vehicle and morphine as per 7-d schedule given in Table 1 (see Additional File) [10]:
Group 1: N. Saline 0.9% (1.0 ml/kg, p. o., twice daily)
Group 2: D. d. (350 mg/kg, p. o., twice daily)
Group 3: Morphine (10 – 100 mg/kg, i. p., twice daily)
Group 4: D. d (350 mg/kg, p.o., twice daily) along with Morphine (10 – 100 mg/kg).
Group 5: D. d. (700 mg/kg, p.o., single dose) 10 h after the last dose of morphine.
Group 6: D. d (700 mg/kg, p.o., single dose) 10 h prior to the first dose of morphine.
Technique
The morphine abstinence syndrome, on which the assessment of physical dependence of morphine was based, consisted of a variety of motor and vegetative signs. The signs of spontaneous abstinence syndrome were recorded 12 h after the last dose of morphine for 30 min in a clear perspex acrylic box of 35 × 22.5 × 13.75 cm. Similar observations were also done in D. d. and Saline treated groups. The 'counted signs' and 'checked signs' were multiplied with the respective 'weighing factors' Table 2 (see Additional File) for evaluation of the severity of abstinence syndrome using a modified methods of Blasig J, et al. and Neil & Sparber [11, 12].
Statistical analysis
Results are expressed as mean ± SEM. Statistical analysis of the data was performed using a non-parametric analysis of variance (ANOVA) and Dunnets Multiple comparison test followed by Friedman test with post-test. P value < 0.05 was considered significant. All statistical procedures were performed with GraphPad software version 3.05 (San Diego California USA, http://www.graphpad.com)