The Preliminary Pharmacy And Pharmacological Researches On The Extract Of Caulis Sinomenii

Objective: To develop the natural ingredients from Caulis Sinomenii, a traditional Chinese herb, which can inhibit opium-dependence and treat the abstinent syndrome. To optimize the best technology through orthogonal design method. To identified Sinomenine, a marked ingredient of sinomenii extract, by means of TLC and to assay the content of sinomenine with HPLC for quality control. The methodology in primary research was evaluated and the qualitative standard of the extract was worked out. The acute toxicity test of the extract for providing a toxicological proof for clinical practice, through models of morphine-dependent rats and mice to determine the effects of sinomenii extract on withdrawal syndrome and to provided a pharmacology basis for Caulis Sinomenii used in clinic.Methods:1. Inspecting test on technology of extracting from Caulis Sinomenii: there are some factors would effect the active ingredient: decocting time, decocting times, the concentration and the volume of ethanol as a vehiculum. Based on the characteristic of Sinomenine which can be dissolved into water and alcohol, we chose different concentration of ethanol. Because the amount of extractive was relative with decocting time and the volume of ethanol, we made decocting time, decocting times, the concentration of ethanol, the volume of ethanol to be the 4 signify factors, and each factor has 3 levels. Therefore we chose the L9 (3⁴) orthogonal design, so the whole experiment was according to the orthogonal design, and each test was repeated 3 times. The content (mg/ml) of Sinomenine was acted the marked index to select the best technical condition for extrative.2. Confirmatory test of extractive technology of Caulis Sinomenii: 3 batches of Caulis Sinomenii extract was verified according to the best technical condition and the content of sinomenine in the extract was detected.3. Qualitative Analysis of Caulis Sinomenii: In accordence with the method of TLC chromatograms (Chinese Pharmacopoeia, 2005, the first part, Appendix VI B), the sampling solution 10μl, the reference crude drug solution 10μl and the reference solution 5μl were used to point on a thin layer silica gel G plates which made of 2% NaOH solution, the upper layer of solution of methylbenzene-methanol-ehtyl acetate-water(2:2:4:1) kept below 10℃was used as the developing agents. The bismuth potassium iodide solution and natrium nitrosum solution were used as color- developing agents in sequence.4. Selection of detecting wavelength: Detected the reference solution and test solution under the chromatograms conditions. Decided the detecting wavelength of Sinomenine according to the ultraviolet.5. Linearity: Accurate 5, 10, 15, 20, 30μl of sampled reference solution were detected by HPLC in triplicate respectively. The standard curve was drawn that used the peak area as vertical coordinate, and the sample volume as horizontal coordinate.6. Precision: The precision was determined with the same sample solution 10μl, injected for 5 times consecutively, and measured the peak area of the Sinomenine.7. Stability: the stability was determined with the same test solution 10μl which was allowed to stand for 0, 2, 16, 18, 22h separately.8. Reproducibility: The reproducibility was determined with the same batch dry extract. Five sample portions of lg each was separately extracted as mentioned in test solution to produce 5 test solutions. 9. Recovery. 5 portions of definite amount of the dry extract (lg), and detected the content of the Sinomenine by HPLC. Added accurately and respectively Sinomenine (2.722mg/ml) lml to these 5 portions samples.10. Contents of Sinomenine in the drug sample: according to the method mentioned in test solution.11. Acute toxicity test: 40 kunming mice (half male, half female) were used randomly divided into 2 groups (n=20): control group and Sinomenii extract group. After fasted for 12 hours (drinking unlimited), the mice of both groups were given normal saline (the same volume) and Caulis Sinomenii extract solution., 2 times a day (9: 00, 18: 00), the toxic reaction in the mice was observed for 14 days after administration. All mice were killed after weighting them at the 15th d. The pathology change of the main organ (heart, liver, spleen, lung, kidney, brain, stomach, etc.) were observed with the naked eyes.12. Pharmacological experiments:(1) The precipitated withdrawal test in morphine dependent rats: The model of morphine-dependent rats was established by dose-increasing administration. 60 SD rats(half male, half female), were used and were subcutaneously injected with morphine, 3 times a day(8:00, 14:00, 20:00). The dosage was 5, 10mg/kg for 4 d respectively, 15, 20mg/kg for other 3d respectively. In 15th day, the rats were randomly divided into 6 groups(n＝10), and received different treatment:â‘ morphine group(morphine 20mg/kg/d, sc, 0.2ml/100g);â‘¡withdrawal model group (saline, ig, 1 ml/100g);â‘¢-⑤three dose groups of Sinomenii extract (Sinomenii extract solution 0.4g/kg, 0.8g/kg, 1.6g/kg, respectively, ig, lml/100g).â‘¥Sinomenine group, (Sinomenine 45mg/kg, ig, 1 ml/100g). After administration 45 min, the withdrawal syndrome was precipitated with naloxone (sc, 4mg/kg) and was recorded in 30 rain. The rat weight was recorded in 60min.(2) The precipitated withdrawal test in morphine dependent mice: The model of morphine-dependent mice were established by dose-increasing administration. 100 Kunming mice (half male, half female), were used and were subcutaneously injected with morphine for 6 days, 2 times a day(8:30, 16:30). The dosage was from 25mg/kg to 150 mg/kg increasing administration to made the morphine dependent model. In 7^th day, the mice were randomly divided into 5 groups(n=20), and received different treatment:â‘ morphine control group(saline, ig, 1 ml/100g);â‘¡-â‘£three dose groups of Sinomenii extract (Sinomenii extract solution 0.8g/kg, 1.6g/kg, 3.2g/kg, respectively, ig, lml/100g).⑤Sinomenine group, (Sinomenine 60mg/kg, ig, lml/100g). After administration 45 rain, the withdrawal syndrome was precipitated with naloxone (sc, 8mg/kg)and was recorded in 30 min. The weight of mice was recorded in 60min.Results:1. The optimum extracting technology of Caulis Sinomenii: The optimum extraction conditions were 8 fold of 70% ethanol under reflux for 2 times, and 2 hours per time.2 In order to verify the optimum extracting technology, dry extract that made from l g crude drug had been determined by HPLC. The result showed that the content of Sinomenine was stable that means the extracting technology is stable too.3 The TLC chromatograms were obtained all samples of Caulis Sinomenii extracted by the optimum extracting technology corresponded in color and position to the spots of the reference substance.4 According to the ultraviolet map of Sinomenine, the wavelength of maximum absorption was 262nm.5 The results showed excellent correlation, and the linear rang was from 2.57μg～15.42μg of Sinomenine.6 RSD (%)=0.99, the result showed great precision.7 The result showed the test solution was stable in 22h after prepared.8 RSD (%)=1.66, the result showed the quality assay of Sinomenine has great reproducibility.9 RSD (%)=1.66, the result showed the quality assay of Sinomenine has great recovery.10 The result showed that content of Sinomenine(C₁₉H₂₃NO₄) in the lg product was no less than 4.4rag.11 The result of acute toxicity test showed that there was no mice died in 14 days when Sinomenii extract was administed with the maximum dosage and capacity, 2 times a day.12 (1) Caulis Sinomenii extract at three tested dosages could significantly decrease the scores of withdrawal syndromes on the rat models of naloxone-precipitated tests and alleviate the weight loss of rats. There were no significant difference between mid-dose or high-dose of Sinomenii extract and Sinomenine group, P＞0.05.(2) The mice were precipitated with naloxone after treated with morphine for 6 days. The mice of model group showed an obvious jumping reaction. The jumping frequency of med-dose, high-dose group of Sinomenii extract and Sinomenine group was obviously less than model group, P＜0.05. There was a significant difference between high-dose group of Sinomenii extract and Sinomenine group, P＜0.05.Conclusion:1 Inspecting test on technology of extracting from Caulis Sinomenii showed that the content of Caulis Sinomenii was stable, and the optimized technology was stable too.2 According to the methodological research, the experiment showed that the method of Sinomenine determination was accurate and had a good reproducibility. This method can be used in Sinomenine determination of Caulis Sinomenii extract.3 The maximum dosage of Sinomenii extract in a day was 64 g/kg. Bw, equal to 914 times of human body. The result showed that the extract was a low toxicity and safe substance.4 Caulis Sinomenii extract could significantly inhibite the withdrawal syndromes and weight loss of morphine-abstinent rats.5 Caulis Sinomenii extract could markedly inhibit jumping reaction and weight loss of morphine-withdrawal mice.