| BackgroundThe genus Isodon is a kind of perennial herbage, semi-shrub of Labiatae. There are about 150 species in the genus, about 30 of which were found to be promising for potential used as traditional Chinese medicine for fever along with detoxication, treatment of splenitis and mobilization of blood circulation, anti-bacteria and diminish inflammation. And some of them have been used as folk medicine for anti-tumor. What’s more, a mass of biological activity researches indicated that abundant diterpenoid compounds obtained from those plants emerged dramatic anti-cancer activity. Isodon neivosus(Hemsl.)C. Y. Wu et H.W.Li, also known as Dayeshezongguan or Lanhuachaihu or Maiyexiangchacai, a perennial herb belongs to Isodon genus of Labiatae, of which the stems and leaves have been used satisfactorily for the treatment of fever and inflammation along with detoxication, acute infectious hepatitis, snakebite and skin itching. Ent-kaurane diterpenoids isolated from Isodon and containing a-methylene cyclopentanone unit were most active towards anti-cancer. These studies have accelerated progress of the use of Isodon plant resources.Nodosin, a pentacyclic 6,7-seco-ent-kaurane diterpenoid with an enmein skeleton extracted from the genus Isodon (Labiatae), was discovered to show the inhibiting and cytotoxic effects on cancer cells in some degree, such as the malignant glioma cells C6 and U87, B16-F10 mouse melanoma cells, neuroblastoma cell line (SHSY5Y), eukemia HL60 cells, human breast cancer cell line MCF-7 and so on.Glioma cells proliferation and growth were obvious selectively inhibited by nodosin. And the IC5o values of nodosin were about 25.81μM and 11.05μM after 24 h treatment in the C6 cells and U87 cells, respectively. In our preceding researches, we found that nodosin exerted the inducing apoptosis effect on glioma cell and inhibited migration and invasion metastasis of glioma cells associated with promoting cell microtubule depolymerization and decreasing the protein levels of MMP-9. We also discovered that nodosin possessed the function of resistance malignant glioma in vivo experiments for the first time, which inhibited tumor growth and reduced the tumor weight in nude mice.To our knowledge, there is still no research about the absorption, metabolism and other pharmacokinetic character of nodosin. For the purpose of providing a meaningful basis in pharmacokinetic for the nodosin development, we carried out a study on the pharmacokinetic of compound nodosin. Based on high performance liquid chromatography-tandem mass spectrometric (HPLC-MS/MS), the method for the determination of nodosin in biological sample was described in this paper, which was used subsequently to study the pharmacokinetics of nodosin in mice and rats.ObjectiveOn the basis of previous research about the nodosin’s function of resistance malignant glioma in vivo and in vitro, this study aims to investigate the absorption, distribution of nodosin in mice and its absorption, excretion in rats, evaluating its pharmacokinetics. First, the experiment developed a simplified, sensitive and specific high-performance liquid chromatography-tandem mass spectrometry assay for the quantification of nodosin in biological samples and then determine the plasma concentration to investigate the pharmacokinetic profiles of nodosin in rats and mice with the method. Secondly, the distribution of nodosin was discussed by determining the concentration in mice brain tissue and hepar, which estimated whether nodosin can penetrate the blood brain barrier. Lastly, the study of excretion was to be acquired by determining the concentration in rats’urine and feces.MethodsIn the experiment, oleanolic acid was used as the internal standard (IS). After chromatographic and mass spectrometer conditions optimized, the study developed a HPLC-MS/MS method for the determination of nodosin in biological samples. In the process of liquid-liquid extraction (LLE) pre-treatment to samples, three key parameters of LLE were considered and optimized:the time durations of vortexing, the amounts of extraction solvent and extraction solvent. According to some reports, factors related to matrix effects and recovery were considerated, thus simplifying the whole sample preparation procedure. Method validation was examined for specificity, precision, recovery and so on.In the animal experiment, after a single intraperitoneal injection of nodosin (1 mg·kg-1) to SD rats and KM mice, biological samples were collected at different time points (periods). And then samples were quantified by HPLC-MS/MS. Pharmacokinetic parameters were directly calculated by using the pharmacokinetic software DAS.3.2.6.Results(1) The study developed a simplified, sensitive and specific high-performance liquid chromatography-tandem mass spectrometry assay for the determination of nodosin in biological samples. The results showed that the linear range of the calibration curve was 3.9～1000 ng·mL-1 (r2>0.99). The lower limit of quantitation (LLOQ) was 3.9 ng·mL-1. The overall mean recoveries of nodosin in biological samples were greater than 74.2%. The precisions of intra-day and inter-day were less than 9.8% and the accuracy ranged from -1.6%to 10.8%. This method could satisfy the analysis of vast samples.(2) The animal experiments determined the absorption, distribution of nodosin in mice and its absorption, excretion in rats, evaluating its pharmacokinetics. Nodosin could not be determined in brain tissue, which explicated that nodosin can’t penetrate the blood brain barrier.1% nodosin was excreted from rat urine and feces in the form of prototype after a single intraperitoneal administration to rats. |
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