Study On The Quality Of Koumine And Koumine Hydrochloride

Koumine is a major alkaloidal constituent of Gelsemium elegans Benth. Our pervious works showed that koumine had significant pharmacological effects for chronic pain and rheumatoid arthritis with low toxicity, which indicates that koumine has a great pharmaceutical potential. As a result, various preparations were prepared for further research. Namely, the quality of koumine is needed to be evaluated. As far as the process of drug research and development is concerned, the law of the People's Republic of China pharmacopoeia 2010 edition has been set up to establish a scientific,reasonable and standard guidance for New drug registration study on chemical drug quality control and impurity research technical guidelines promulgated by the CFDA to study on the quality of koumine, the acute toxicity and analgesic activity of related substances. The main contents are as follows:1. Preparation of koumine and koumine hydrochlorideWe extracted the total alkaloids of GEB from the roots and stems of Gelsemium elegans Benth with alcohol, the extraction rate was 0.48%, Preparative separation of koumine from the total alkaloids of GEB was used, p H-Zone-Refining.Preparation of different batches of koumine hydrochloride,koumine was dissoluted by acid, and precipitated by alkali then we got koumine. This part of the study has 3 batches of koumine(production respectively were 2.2 g, 2.1 g, 2.4 g; batch number were KM201504,201505, 201312) and 5 batches of Koumine hydrochloride(production respectively were 5.6 g,4.1 g,4.4 g,3.6 g,4.1 g; batch number were KM201308,201309,201310, 201311, 201310), their UPLC assay purity are all more than 98.5%, for the following study.2.Study on the quality and preliminary stability of koumine and koumine hydrochloride.2.1 Study on the quality of koumine, koumine hydrochloride and the establishment of quality standard.Establishing the UPLC detection method of koumine and koumine hydrochloride,which can be used to study the quality of koumine and kounine, is stable and reliable.Through that way did we investigate three batches of koumine and 3 batches koumine hydrochloride's character(appearance, hygroscopicity, solubility, melting point,specific rotation, absorption coefficient), identification method(UV-VIS,UPLC, IR),examine(related substances, clarity and color of the solution, p H value, moisture) and content determination(koumine content), then set up the laboratory quality standard as follows:2.2Preliminary stability study of komine and koumine hydrochlorideThe results of influential factors test showed that at 60? for 10 days or at 25 ?,relative humidity 90% for 10 days, the character,content of related substances,melting point,clarity and color of the solution had no significantly change,which suggested that preparation stability was good in relative humidity of 90% at room temperature,but it had slight hygroscopicity,and illuminated in the light of 4000 LX for 10 days,the color of koumine change from white to faint yellow; which informed us that koumine should keep in dark place. We conducted the accelerated test on the basis of influential factors test, result of which showed that at the temperature of 40? and relative humidity of75% for 6 months, the character,content,related substances,melting point,clarity and color of the solution of koumine also had no significant change. It provided experimental basis for storage conditions of koumine.3.Separation of koumine of relevant material and structure identificationDeterminationed K values of related substances in different solvent system by UPLC, finally choosed chloroform:methanol:0.2 M hydrochloric acid water(4:2:2)system.Preparated the solvent system, the related substance 1 and 2 that were isolated from koumine, and compared the reference substance with them by UPLC. Meanwhile,we detected Ms and NMR spectrum of the two relevant material then compared them with the relevant literature to demonstrate that the chemical structure were gelsenicine and gelsevirine.4. Acute toxity and analgesic activity of related substancesIn order to define the acute toxity of related substance 1 and 2 in mice by gastric.LD50 was determined.As a result LD50 of substance 1 and 2 were 0.075 mg/kg and 33.648 mg/kg, and 95% confidence interval were 0.050--0.113 mg/kg and23.596--49.642 mg/kg. The analgesic effect in mice of the total alkaloids of GEB,koumine and related substance 2 were obvious by writhing response and hot-plate pain response.But the related substance 1 had no effect,The study on the quality laid the foundation for the laboratory research.In summary: In such a study have we successfully prepared komine and koumine hydrochloride, the production to reach small scale, and the purity of drug more than98.5%. Then we also carried out the preliminary stability study, the stability of komine and koumine hydrochloride, which is good. It provides the establishment of its storage conditions with laboratory basis, establishes komine and koumine hydrochloride laboratory quality standards through quality research. In the meantime, this study has confirmed that the related substance of komine and koumine hydrochloride were gelsenicine and gelsevirine, and the later of which determined the acute toxicity and analgesic activity of gelsenicine and gelsevirine on mice, which lay a laboratory foundation for quality control of komine and koumine hydrochloride.