Chemical Constituents And Pharmacology Of Chinese Medicine Polyporus Study

Polyporus umbellatus, called in Chinese "Zhuling", is prepared from the dried sclerotia of P. umbellatus (Pers.) Fries (Polyporaceae). It is one of the most important traditional Chinese medicines (TCM), and is distributed and used in Asia (mainly in China and Japan), Europe and North America. It has been used as a folk medicine in China for more than 2500 years. P. umbellatus was first described in a famous Chinese medical book, the Shen Nong Ben Cao Jing. Later, it appeared also in other Chinese traditional medicine books, such as Zhong Hua Ben Cao, and Shang Han Lun, ect. Presently, P. umbellatus officially listed in the current Chinese Pharmacopoeia. P. umbellatus is used currently in China as diuretic and antibacterial drug, the diuresis being its main pharmaceutical function. Many medical prescriptions contain P. umbellatus, for example, Zhuling Tang, Zhuling Wan, Zhuling San, Wuling San, Fenxiao Tang, Yinchenwuling San, etc. These prescriptions showed excellent efficacy in clinical application, and no report on its toxicity could be found in the literatures.The thesis consists of six chapters altogether. The second chapter presents the reviews on steroidal constituents, non-steroidal steroidal constituents, pharmacology and references of Polyporus umbellatus. The second chapter shows the isolation and identification of all kinds of compounds from Polyporus umbellatus.24 compounds were purified and identified, which concern steroids, triterpenoid, alkaloids anthraquinones phenylpropanoids and cermaide. One compound was proved to be new one and 5 compounds were isolation from Polyporus umbellatus for the first time. The third chapter studies the determination of the main steroidal constituents in Polyporus umbellatus by HPLC methods for quality control of Polyporus umbellatus. The forth chapter studies the diuretic constituents of n-hexane, ethyl acetate, n-butanol and water extracts of Polyporus umbellatus and to evaluate their diuretic activity. ergosta-4,6,8(14),22-tetraen-3-one (ergone), ergosterol and D-mannitol display high diuretic activity. The forth chapter studies quantitative determination of ergone and its metabolite in rat plasma, feces and urine by HPLC-MS/MS, HPLC-APCI-MS", HPLC-FLD and HPLC-UV methods and its application to pharmacokinetic studies. The sixth chapter studies the anticancer components from methanol extracts of Polyporus umbellatus using bioactivity-guided method of Human hepatocellular liver carcinoma cell line (HepG2) and its apoptosis mechanism. The results show the steroidal components have strong cytotoxic activity and Ergone induces G2/M cell cycle arrest and apoptosis in human hepatocellular carcinoma HepG2 cells.Chapter 1. Advances in the Study of Chemical Constituents and Bioactive Constituents of Polyporus UmbellatusThe review covers steroidal constituents, non-steroidal steroidal constituents, pharmacology and references of Polyporus umbellatus.Chapter 2. Study on Chemical Constituents of Polyporus UmbellatusPolyporus umbellatus is often used diuretic medicine. As reported previously, the chemical constituents of Polyporus umbellatus mainly involve polysaccharides and steroids, and they are often considered as important bioactive substances. Polysaccharides were developed to an antitumor medicine and have been proved to be efficacious in clinical application. For this reason Polyporus umbellatus polysaccharide has attracted much attention of medicinal chemists in the world. So far, the studies of Polyporus umbellatus have been focused mainly on its chemical component, pharmacology, efficacy and application. Traditional Chinese medicine and contemporary pharmacological studies demonstrated that Polyporus umbellatus had activity of diuresis and was effective for treatment of some kidney diseases. However the chemical structure of diuretic bioactive components has been ambiguous. Polyporus umbellatus was collected and isolated from Taibai mountain of Shaanxi Province in order to find diuretic constituents.24 compounds were purified and identified, which concern steroids, triterpenoid, alkaloids anthraquinones phenylpropanoids and cermaide. One compound was proved to be new one and 5 compounds were isolation from Polyporus umbellatus for the first time.Chapter 3. Determination of Steroids Constituents from Polyporus Umbellatus by HPLC MethodsIn order to increase the content determination of Polyporus umbellatus in the "Chinese Pharmacopeia", the chemical constituents of P. umbellatus were separated and analyzed in our laboratory by repeated silica gel column chromatography and HPLC-UV methods. Ergosterol was found as a major characteristic constituent, and its content, highest in Polyporus umbellatus. Ergosterol, is regarded as a marker compound. The content determination of ergosterol in P. umbellatus is now officially listed in 2010 edition of the "Chinese Pharmacopeia" for quality control and evaluation.Ergone was a diuretic bioactive component, its content was determinated by HPLC-UV method. Because of very low content, the content of ergone could not be accurately determinated by common analytical method. In this experiment, a HPLC-FLD approach was applied for quantitative analysis of ergone in P. umbellatus from different habitat districts. The reliability of the method was further verified by LC-APCI-MS/MS. The method was adaptable for evaluation of active compounds in Polyporus umbellatus.One essential difference of traditional medicines from chemical drugs is that their therapeutic effects are due to the contribution of multi-components, not only the major ones. Polyporus umbellatus therapeutic effects are also due to the contribution of multi-components. So using only one component in Polyporus umbellatus for quality control is not comprehensive and systematic. The quantitative evaluation of the active components in Polyporus umbellatus has been a challenge in phytochemistry. It is necessary to identify the major compounds and their distribution in Polyporus umbellatus, in order to elucidate the material foundation of therapeutical effect of Polyporus umbellatus.As a result of our study, eight steroids have been isolated from Polyporus umbellatus by column chromatography. Their chemical structures were characterized by spectroscopic techniques. Here, we would like to report a simple, fast and sensitive high-performance liquid chromatography-atmospheric pressure chemical ionization-mass spectrometric detection (HPLC-APCI-MS) method for simultaneous separation, qualitative and quantitative determination of eight major steroids in ten batches of Polyporus umbellatus from different habitat districts for the first time. At the same time, the HPLC-APCI-MS method was compared with high-performance liquid chromatography-photodiode array detector (HPLC-PAD) and high-performance liquid chromatography-evaporative light scattering detector (HPLC-ELSD). The HPLC-APCI-MS method can provide low limit of detection, high selectivity and high sensitivity in comparison with HPLC-PAD and HPLC-ELSD method. The contents of eight major steroids were investigated to reveal their differences among ten batches of Polyporus umbellatus. The method was adaptable for analysis of steroid compounds and also formed a characteristic pattern of Polyporus umbellatus. This study provided more comprehensive analysis method for further research of the mechanism and the accumulation of its secondary metabolites. Chapter 4. Pharmacology of ergone diuretic and prevention effect of chronic renal failureIt is thought that Polyporus umbellatus, among the crude drugs, possesses a relative diuretic effect and the ability to reinforce the functioning of water pathways capable of drain dampness. It was also described that it promotes urination and leaves out dampness, the problems caused by stagnance of dampness such as edema, scanty urine, vaginal discharge, cloudy painful urinary dysfunction, as well as jaundice and diarrhea. The n-hexane, ethyl acetate, n-butanol and water extracts of P. umbellatus were tested by diuretic experiment of normal rats in metabolic cage. The ergone, ergosterol and D-mannitol were isolated and identified from Polyporus umbellatus by bioactivity-directed method. They were main diuretic bioactive components in Polyporus umbellatus.The experiment was performed by adenine-induced chronic renal failure in rat in order to find treatment medicine of chronic renal failure. The total protein, albumin, globulin, total cholesterol, triglyceride, urea, creatinine and blood routine were determinated. The results showed that ergone can increase hemoglobin content, decrease urea and creatinine content significantly, markedly decrease ColⅠ,ColⅢand TGF-(31, and improve renal function. Ergone can treat adenine-induced chronic renal failure in rat.Chapter 5. Pharmacokinetics of ergone from Polyporus UmbellatusBase on ergone pharmacology, HPLC-MS/MS, HPLC-APCI- MSn, HPLC-FLD and HPLC-UV method was developed for identification and quantification of ergone and its metabolite in rat plasma, urine, and feces. Deproteinization with methanol or acetone was chosen for it provided a clean supernatant with high extraction recovery. Additionally, the simple extraction procedure made it feasible for quantitative analysis. HPLC-MS/MS and HPLC-FLD methods were applied for the quantification of ergone. The results indicated that the two methods showed high recovery, good precision and accuracy. The results provide reliable data in clinic application for Polyporus umbellatus.Chapter 6. Cytotoxicity Components from Polyporus Umbellatus and ergone induces G2/M cell cycle arrest and apoptosis in HepG2 cells The steroids ergone, (22E,24R)-ergosta-7,22-dien-3β-ol,5α,8α-epidioxy-(22E,24R)-ergosta-6,22-dien-3β-ol, ergosta-6,22-dien-3β,5α,6β-triol and polyporusterone B against HepG2 were isolated from Polyporus umbellatus by bioassay-guided approach. They showed potent anticancer activity against HepG2 cells. Ergone displayed remarkable anticancer activity against HepG2, Hep-2 and Hela cancer cells, of which, HepG2 cells were the most sensitive. Meanwhile, the cytotoxic effects of ergone on normal human cells (HUVEC) were smaller than on cancer cells. The results showed ergone had more selective cytotoxic activity against cancer cells than normal cells. The result showed the steroidal constituents of Polyporus umbellatus have strong cytotoxic activity and Ergosta-4,6,8(14),22-tetraen-3-one induces G2/M cell cycle arrest and apoptosis in HepG2 cells in a caspase-dependent manner.