Study On Analysis In Vitro And Disposition In Vivo Of Steroidal Saponins In Paris Polyphylla

Paris Polyphylla, the dried rhizome of Paris polyphylla Smith Var. yunnanensis(Franch.) Hand-Mazz or Paris polyphylla Smith Var. chinensis (Franch.) Hara, namedChonglou in Chinese, has been widely used in Chinese folk medicine for a long time. Itwas first recorded in―Shennong Herb‖named as Zaoxiu. As a typical herbal medicine,Paris Polyphylla is usually used to treat fracture, cough, abscess, hemostasis, snake bite,convulsion in clinical. Extensive pharmacological studies indicated that the methanolic ex-tract and aqueous extract of Paris Polyphylla had potential anticarcinogenic activity, im-munity adjustment, analgesia and anti-inflammation. And steroidal saponins are generallyregarded as its main bioactive ingredients. Therefore, in this study, based on modern ana-lytical techniques, such as high-performance liquid chromatography time of flight massspectrometry (HPLC-TOF/MS), liquid chromatography ion trap mass spectrometry(HPLC-IT/MS), liquid chromatography mass spectrometry (HPLC-MS), liquid chroma-tography tandem mass spectrometry et al, we developed several methods and strategies toqualitative and quantitative study steroidal saponins in in vivo and in vitro, and preliminaryscreening of potential pharmaceutical active components.1The anti-cancer activity study of steroidal saponinsIn the investigation, we selected polyphyllin I and polyphyllin VI which are two highlevel steroidal saponins in paris polyphylla for pharmacodynamic study. And found thesetwo steroidal saponins have a strong cytotoxic activity. For Hepatoma cells HepG2andSMMC-7721, IC50value of polyphyllin I are both about3μM, and IC50value ofpolyphyllin VI are about6μM. The results indicated that the activity of polyphyllin I isbetter than VI, probably because there is a Hydroxy-substituted in C-17of polyphyllin VIwhich reduced its Cytotoxicity. Besides, a classic MTT method and optimized CCK-8method were chosen as the detection methods. CCK-8method have more significant ad-vantages than MTT method as follows, Formazan produced in CCK-8is water solublesubstance, which simplified the Experimental Procedures for no addition of FormazanDissolving agents, and because of its high sensitivity and stablity, a large amount of sam-ples can be detected in a shorter time.2Study on analysis in vitro of steroidal saponins in Paris PolyphyllaIn the present study, we developed a novel analysis method using high-performance liquid chromatography electrospray ionization time of flight mass spectrometry(HPLC/ESITOFMS) and ion trap mass spectrometry (HPLC/ITMS) for separation andcharacterization of steroidal saponins in Paris Polyphylla extract. As the result, with theadditional retention time and accurate mass measurement of the authentic standards, fivesteroidal saponins in Paris polyphylla extract were unambiguously identified. A formuladatabase of known steroidal saponins in Paris polyphylla was established, against whichthe other30steroidal saponins were identified effectively based on the accurate extractmasses and formulae acquired by HPLC/TOFMS. In addition, multi-stage mass spectrom-etry was introduced to obtain various characteristic fragmentation behavior to distin-guished the3groups of isomers.3Study on disposition in vivo of steroidal saponins after administration of Parispolyphylla extractTo study the absorption, distribution, metabolism and excretion in vivo of the mainsteroidal saponins in Paris Polyphylla, a novel LC-MS/MS method was developed for thesimultaneous quantification of the main steroidal saponins in rat plasma and the establishedmethod validation was carried out according to the USA Food and Drug Administrationbioanalytical method validation guidance. The method was demonstrated to be rapid, sim-ple, sensitive and accurate for the simultaneous determination of polyphyllin I, polyphyllinII, Polyphyllin VI, Polyphyllin VII, dioscin and gracillin with small plasma volume. Allsamples were found to have no interference from endogenous substances at the respectiveretention position of the steroidal saponins. And the intra-and inter-day precisions (RSD%)were less than13%and the average extraction recoveries were ranged from85.%to97.0%for each analyte. Six steroidal saponins was proved to be stable during sample storage,preparation and analytical procedures. Additionally, the method was successfully appliedto a pharmacokinetic study of steroidal saponins after oral administration of Parispolyphylla extract to rats. The pharmacokinetics showed a long half-life for all six steroidalsaponins, which were almost more than10hours, indicating the steroidal saponins have along residence time in the body, and a slow excretion. The drug plasma concentration wasalso very low, although a high dose was given, revealed the steroidal saponins have an ex-tremely low oral bioavailability. Generally, oral bioavailability is affected by several fac-tors including solubility, intestinal microorganisms, intestinal permeability and first-passextraction. In which one or more factors affect the oral bioavailability of steroidal saponins are need further investigation.4In vitro metabolism study of steroidal saponinsHL-7702(Human liver cells) was selected for in vitro incubation because it keeps al-most all the function of human normal liver cell and can proliferate largely. The in vitroincubation experiments of six steroidal saponins Polyphyllin I, Polyphyllin II, PolyphyllinVI, Polyphyllin VII, Dioscin and Gracillin indicated that they can all be eliminated in livercells, and the steroidal saponins which aglycone type is diosgenin are eliminated fasterthan the pennogenin. The concentration of steroidal saponins in cells were very high afterbroken by Ultrasonic method. Therefore, steroidal saponins may be metabolized in livercells or just be absorbed into cells or be adsorpted on cell membrane. We also choose oneingredient in diosgenin and pennogenin respectively for liver microsomes incubation ex-periments and verify activity of incubation system by testosterone as positive drug. Theresults show that testosterone can be metabolized in incubation system entirely and quickly,which indicates high activity of the incubation system. However, the contents of two ste-roidal saponins have no significant change after be incubated for two hours. The resultsshow that steroidal saponins metabolize stably in liver microsomes, which may be causedby lack of related metabolizing enzymes in liver microsomes and indicate that when incu-bated with normal liver cells, steroidal saponins was just absorbed into cells or adsopted oncell membrane rather than biotransformation.