| Flammulina velutipes, a very popular edible mushroom, is rich in carbohydrates, protein, dietary fiber and minerals nutrients. In recent years, it has been reported that proteins and polysaccharides isolated from F.velutipes had a variety of biological activities such as antiviral, antitumor, immunoregulation and liver protection. In addition, other small molecules including flavonoids, terpenes, fatty acids, sterols and nucleoside, have also been discovered in F. velutipes. The current reports on the biological activities of these small molecules were mainly focused on the aspect of anti-bacterial and anti-oxidation.Ergosterol, one of the main components of the sterols in F.velutipes, has been proved to have a significant antitumor effect. However, as a phytosterol, ergosterol is very difficult to dissolve in water, resulting in the extremely low bioavailability in vivo. Therefore, it is desirable to improve its solubility and bioavailability.So far, few reports have been published to report the research work that sterols from F.velutipes enjoyed a significant antitumor activity and the trials to improve the bioavailability.As one of the most serious diseases hazarding to human health and life, tumor has become one of the top considerations of many experts around the world, who have been looking for all kinds of drugs and therapies solicitously to treat this disease. In this work, it is the first time around the world to use high-throughput screening model——lipid rafts chromatography system based on a kind of receptors of tyrosine kinase inhibitors to obtain substances from F.velutipes with antitumor effects. The substances are the petroleum ether extract and ether extract from F.velutipes. Further study showed that the major components of these two extracts were fatty acids and sterols. In vitro experimental results demonstrated that they both possessed good antitumor activity. However, the effective application of sterols has been limited by the poor water solubility, thus it is urgent to develop methods to improve its solubility and bioavailability. Nanoparticle drug delivery system is an effective way to enhance the solubility and bioavailability of poorly water-soluble drugs. In this experiment, three different kinds of sterols nano-preparations were prepared. Furthermore, these three nano-preparations were administrated to mice by three different routes, and in vivo pharmacokinetics as well as the tissue distribution and excretion were then studied. The results indicated that all the three nano-preparations could significantly improve the water solubility and bioavailability of sterols. This work provided a foundation for the development of sterol as a new drug. The main research work was listed as follows:1. The model of high-throughput screening--lipid rafts chromatography. According to the model of high-throughput screening based on the receptors of tyrosine kinase inhibitors——lipid rafts chromatography, it has been found that sterols and fatty acids extract had antitumor activity. MTT assay was used to examine the proliferation inhibition of the two extracts (sterols and fatty acids) on several different tumor cell lines, such as cultured human hepatoma cell line HepG2, gastric adenocarcinoma cell line SGC, glioma cell line U251, lung adenocarcinoma cell line A549, cervical carcinoma cell HeLa and colon cancer cell line Lovo. The results showed that both sterols and fatty acids exhibited strong cytotoxicity against the human tumor cell lines (HepG2,SGC,U251,A549), and weak cytotoxicity against HeLa and Lovo. The IC50 proliferation inhibition of sterols in vitro on HepG2, SGC, U251 and A549 were 9.29,11.99,23.85 and 20.44μg·mL-1. The IC50 proliferation inhibition of fatty acids in vitro on SGC was 69.91μg·mL-1. The chemical components of the two extracts have been separated and analyzed by gas chromatography mass spectrometry (GC-MS). The results demonstrated that the sterols had two major components (ergosterol and 22,23-dihydroergosterol) and two minor components (Ergosta-5,8,22-triene-3-ol and Ergost-8(14)-ene-3-ol), and the fatty acids contained 19 kinds of ingredients, with the hexadecanoic acid, 9-octadecenoic acid,9,12-octadecadienoic acid, and 9,12,15-octadecadienoic acid in majority. Additionally, the two major components (ergosterol and 22,23-dihydroergosterol) in the extract of sterols were separated and prepared by high performance liquid chromatography (HPLC),2. The establishment of the reverse phase-high performance liquid chromatography (RP-HPLC). The methodology RP-HPLC was established to determine the contents of ergosterol and 22,23-dihydroergosterol in F.velutipes fruiting bodies. The analysis was carried out on an HPLC system equipped with Inertsil ODS-SP column (250 mm×4.6 mm,5μm) eluted with the mobile phase consisting of 98%methanol. The flow rate was 1.0 mL·min-1 and the detection wave length was set at 282 nm. Ergosterol and 22,23-dihydro-ergosterol showed a good linear relationship within 0.582～72.768μg·ml-1 and 0.586～73.248μg·ml-1, respectively. The results indicated that the contents of ergosterol and 22,23-dihydroergosterol in dried F.velutipes fruiting bodies were 0.30% and 0.072%, respectively.3. Preparation and characterization of sterols nano-preparations. In order to improve the solubility of sterols, three different kinds of sterols nano-preparations (oil-in-water microemulsion, liposomes and nanomicelles) were prepared, and their basic properties were then investigated in this work. The encapsulation rate of the microemulsion was (81.1±1.2)%, with an average particle size of 22.9 nm and a good stability. In the case of liposomes prepared in this work, the average particle size was 108 nm with the polydispersity index of 0.151, and the encapsulation rate was (71.3±0.1)%. For the micelles, the average particle size was 115.6 nm with the polydispersity index of 0.029, and the encapsulation rate was (76.6±0.2)%. In a word, the nano-preparations produced in this work possessed small sizes and high encapsulation rates.4. Establishment of HPLC method for the determination of sample concentration in vivo. A HPLC method were developed for the first time to simultaneously determine the concentration of ergosterol and 22,23-dihydroergosterol in plasma, tissue samples and fecal samples. The analysis was carried out on a HPLC system equipped with an Inertsil ODS-SP analytical column (250 mm×4.6 mm,5μm). A 96% methanol as the mobile phase for plasma, heart, liver, spleen, lung, kidney, brain and fecal samples, and 93% methanol for stomach homogenate. The flow rate was 1.0 mL·min-1. The samples were detected using an UV detector at the wavelength of 282 nm, with the detection sensitivity of 0.02 AUFS. The results demonstrated that this method enjoyed high specificity and accuracy, and other indicators such as the recovery rate and stability of the frozen samples met the requirement for in vivo drug analysis.5. The study of pharmacokinetic. In the study of pharmacokinetic, three different nano-preparations of both ergosterol and 22,23-dihydroergosterol were given to rats by three different routes of administration. The raw drug (sterols) was used as the control. The results were as follows:(1) After oral administration, AUC0-24 of ergosterol in microemulsion, liposomes and micelles were 2.6-,1.6- and 1.5-fold, respectively, of that of the raw drug. The AUC0-24 of 22,23-dihydroergosterol in microemulsion, liposomes and micelles were 4.5-,2.5- and 2.8-fold increased respectively, compared to that of the raw drug. (2) After intraperitoneal injection, the AUC0-24 of ergosterol in microemulsion, liposomes and micelles 47.5,4.3 and 8.4-fold increased, respectively, compared to that of the raw drug. The AUC0-24 of 22,23-dihydro-ergosterol in microemulsion, liposomes and micelles were 14.46,1.47, and 2.11μg h·mL-1, but 22,23-dihydroergosterol in raw drug in the blood could not be detected. (3) After intravenous injection, the elimination half-life and AUC0-24 of ergosterol in microemulsions, liposomes and micelles in the plasma were 0.75,0.86 and 0.72 h, respectively, and the AUC0-24 of ergosterol in the nano-preparations above were 101.16,91.83 and 124.10μg·h·mL-1, respectively. In the case of 22,23-dihydroergosterol, the elimination half-life in microemulsions, liposomes and micelles in the plasma were 0.85,0.85 and 0.75 h, respectively, and the AUC0-24 of the nano-preparations above were 50.60,43.22 and 56.16μg·h·mL-1, respectively. These results indicated that nano-preparations prepared in this study could remarkably enhance the bioavailability of sterols in rats.6. In vivo distribution. To study the distribution of samples in rats, the raw drug (sterols) for the control, three different sterols nano-preparations of both ergosterol and 22,23-dihydroergosterol were given administrated to rats by three different routes of administration. The concentration of ergosterol and 22,23-dihydroergosterol in the tissues and excrement were then determined, respectively. The results were listed as follows. (1) one hour after oral administration, drugs could be detected in most tissues, and mainly concentrated in the blood-flow-sufficient tissues including liver, spleen, lung and kidney. Due to the poor absorption of the raw drug, there was no evidence of the raw drug in most organs 1 h after administration. Interestingly, there was little or no distribution of samples in the heart and brain tissues. Because of the tmax of drug oral absorption is between 5-8 h, drug concentrations were generally low in the tissues. There were a large number of drugs in the feces at 4 h post administration in the micelles and the raw drug group. (2) one hour after intraperitoneal injection, except for the heart and brain tissues, the drugs could be detected in the rest of the tissues. In liposomes and micelles groups, the highest drug distribution was in the liver, followed by spleen, lung, kidney and stomach. The content of the microemulsion group in each tissue was lower than the two groups mentioned above because of the greater tmax of microemulsion via intraperitoneal injection. For the raw drug group, there were very low concentrations in most tissues, but a high content in excrement. (3) one hour after intravenous injection, in the nano-preparations groups, drug distributed mostly in the liver, followed by lung and kidney. In the raw drug group, there was a high distribution in the kidney. All the results showed that the three kinds of sterols nano-preparations could enhance the distribution of samples in liver, spleen and lung. |
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