Study On Preparation Of Oridonin And Polysaccharide From Rabdosia Rubescens And Their Antineoplatic Effects On Bladder Tumor

Bladder cancer is the most common malignancy of urinary system in China.Ninety percent of bladder cancer is transitional cell carcinoma (BTCC), of whichseventy to seventy five percent is superficial bladder cancer. Of superficial bladdercancer, ninety five percent is high-grade BTCC. After surgery, forty percent tends torecur in two years.with a long-term recurrence of almost 100%. Of the superficialBTCC, ten percent will progress to be invasive. Of the high-grade superficial BTCC,the short-term recurrence is up to sixty to ninety percent if not interfered. Because ofthat, intravesical instillation of anticancer agents should aim at clearing tumor,preventing recurrence and delaying progression of tumor.Although intravesical administration of Bacillus Calmette-Guerin (BCG) has beenaccepted as the most effective therapy for superficial bladder cancer and carcinoma insitu of the bladder, thirty to forty five percent of patients never respond to BCGtherapy. Furthermore, long-term remission (more than five years) is only achieved infifty percent of patients. Toxicity associated with the BCG therapy is frequent withoccurrence of severe adverse effects in five percent of patients and life-threateningcomplications in 0.5 percent of patients. Patients hardly accept the regular instillationperiod because of severe cystitis and complications. It's reported that the percentageof patients who kept accepting regular instillation is not more than nineteen percent.So, it's necessary to explore a new agent acceptable by patients for intravesicalinstillation with better effects and fewer complications.Nowadays, research on antineoplastic agents has been highlighted by naturalproducts. Rabdosia rubescens is a traditional Chinese herb with a variety of pharma-cological effects, especially anticancer effects. Oridonin and rabdosia rubescenspolysaccharide (RRP) are the two important ingredients which exhibit remarkable inhibitory effects on many tumor cell lines. Clinical reports have demonstrated thatcoarse extract ofrabdosia rubescens can prevent recurrence of bladder cancer.In this dissertation, oridonin and RRP extraction was studied on at first. Afteralcohol back-streaming and separation by silica gel column chromatography, oridoninof which the purity is ninety eight percent was obtained. After water-extraction withalcohol-precipitation, RRP was obtained. Second, we studied the antineoplasticeffects of oridonin and RRP on bladder cancer cell lines, MB49 cells, EJ cells andT24 cells. After establishing an orthotopic murine BTCC model successfully, wefinally studied the effects of oridonin and RRP on murine BTCC and its mechanism.SectionⅠExtraction and Separationof Oridonin and Polysaccharide from Rabdosia RubescensObjective: To prepare oridonin and polysaccharide from the traditional Chineseherb, Rabdosia rubescens, for the next experiments. Methods: Total diterpenoidswere obtained by alcohol back-streaming. Oridonin was separated by silica gelcolumn chromatography, assayed by high performance liquid chromatography.Polysaccharide was obtained by water-extraction, precipitated by alcohol, andpurified by organic solvents. Results: Extraction rate of oridonin was 0.22 percent(2.2/1000) and the purity of oridonin was 98 percent. Extraction rate ofPolysaccharide was 4.05 percent (8.1/200). Conclusions: The alcohol back-streamingis a better method for extraction of oridonin. The water extraction withalcohol-precipitation is a practical method for preparation of RRP.SectionⅡStudyon Antineoplastie Effects of Oridonin and Rabdosia Rubescens Polysaccharide on Bladder Cancer Cell LinesObjective: To study the antineoplastic effects of oridonin and rabdosia rubescenspolysaccharide (RRP) on bladder cancer cell lines, MB49 cells, EJ cells and T24 cellsin vitro. Methods: The inhibitory rate of the cells was measured by MTT assay. Cellapoptotic rate was detected by flow cytometry (FCM) using Annexin V-FITC andpropidium iodide (PI) detection kit. Morphology of cell apoptosis was observed byHoechst 33258 fluorescence staining and electron/light microscopy. DNA fragmen-tation was assayed by agarose gel electrophoresis. Caspase-3, Caspase-8 andCaspase-9 were assayed with colorimetric assay kits. Bcl-2 and bax expressions weredetected by Western Blot. Telomerase was detected by PCR-TRAP-Silver staining.Results: Oridonin and RRP could inhibit the growth of MB49 cells, EJ cells and T24cells and cause apoptosis significantly. The suppression was both in a time-anddose-dependent manner. IC50 at 24 hours is 16μmol/L in MB49 cells, 25.6μmol/L inEJ cells and 27.2μmol/L in T24 cells respectively. Combined administration oforidonin and RRP could be synergetic in EJ cells and T24 cells. Marked morpholo-gical changes of cell apoptosis including condensation of chromatin and apoptosisbody were observed clearly. Oridonin could down-regulate the Bcl-2 expression andup-regulate the Bax expression of MB49 cells, EJ cells and T24 cells. Oridonin coulddecrease the telomerase activity of MB49 Cells. Oridonin could increase the activityof Caspase-3 and Caspase-9 of the abovementioned 3 cells, and increase the activityof Caspase-8 of EJ cells and T24 cells, but not of MB49 cells. Conclusions: Oridoninand RRP, especially oridonin, have apparent inhibition and apoptosis-inducing effectson MB49 cells, EJ cells and T24 cells. Low concentration of oridonin mainly inducesapoptosis, while high concentration of oridonin may induce necrosis in bladdercancer cell lines involved. Oridonin may induce apoptosis by activating Caspase-3through both Fas/FasL-mitochondria and Apo 2L/TRAIL-Caspase-8 pathways in EJcells and T24 cells, but only via Fas/FasL-mitochondria pathway in MB49 cells. SectionⅢStudy on Intravesical Instillation of Oridonin andRabdosia Rubescens Polysaccharide for Murine Bladder CancerObjective: To establish a model of orthotopic murine bladder cancer, study theeffects of intravesical instillation of oridonin and RRP on murine bladder cancer andobserve the morphological changes in bladder, heart, liver, spleen, kidneys and lungs.Methods: Orthotopic C57BL/6 murine bladder cancer model was established byintravesical instillation of MB49 cells after argentine nitrate cautery. The diagnosis ofbladder cancer was made by routine pathology and electron microscopy. Ten daysafter instillation of MB49 cells, mice were divided into 4 groups, 15 mice in eachgroup. 100 microliters of PBS, oridonin, RRP, combined administration of oridoninand RRP were instilled intravesically in different group respectively. Afterwards,instillation was given in each group every 5 days, 4 times in all. One week after that,mice were all sacrificed. Blood subtypes of lymphocyte were detected by FCM.Bladder, heart, liver, spleen, lungs and kidneys were anatomized out for pathology tocalculate the cure rate and observe morphological changes. TUNEL detection wasmade in tumor-loaded mice. Results: Tumor-taking rate was 96.7 percent in ouranimal model. There were no significant differences in lymphocyte subtypes betweencontrol group and experiment groups. The cure rate in the combined administrationgroup was 93.3 percent, which was higher than those both in oridonin group (80percent) and in RRP group (60 percent), of which differences were significant. Therewere no apparent degeneration, necrosis, inflammatory leukocyte infiltration andfibrosis in the abovementioned viscera. TUNEL detection showed that there wereapparent apoptosis cells in each group. The closer the tumor surface approaches, themore apparent the apoptosis is. Conclusions: The orthotopic C57BL/6 murinebladder cancer model established by instillation of MB49 cells after argentine nitrate cautery is practical for animal experiment of instillation therapy for bladder cancer.Oridonin and RRP have apparent antineoplastic effects, of which mechanism may beinduction of apoptosis. After instillation of oridonin and RRP, heart, liver, kidneys,lungs and spleen are safe without damages.