| Leukemia is a malignant disease of hematopoietic tissue, also known as "blood cancer. " According to the World Health Organization statistics,the whole world has 300,000 new cases to happen every year. The incidence rate of leukemia's disease below 35 years old occupies sixth in all kinds of tumor in various local places which is the highest of the malignant tumor.The proliferation of the leukemia cells in the leukemia patients'marrow, the liver, the spleen, the hemopoietic organs and so on that take the serious influence in the hemopoietic tissue, the skin mucous membrane, the heart involucrum, the spleen, the stomach and the central nervous system and so on, which cause the varying degree hemorrhage, anemia, the infection to give off heat that threaten people's health and the safety. The method currently used mainly for treatment of leukemia is chemotherapy, radiation therapy, targeted therapy and traditional Chinese medicine treatment and so on. The first three treatments mainly rely on chemical drugs, even though it can kill cancer cells fast, pointed and the function is obvious, but they have big toxic side effects and the high drug resistance, they will reduce the body's immune system and bring more serious physical harm and the heavy financial burden to patients. The Traditional Chinese Medicine is rich in resources, it treats the leukemia mainly through itself or the effective component. They can inhibit the proliferation of leukemia cells, induced differentiation and apoptosis of leukemia cells to achieve the effect. And it can reduce the toxic side of chemotherapy and eliminate residual leukemia cells in vivo. It plays a preventive role in the recurrence of the disease. Therefore, the important way to improve the treatment of leukemia is to research and develop the traditional Chinese medicines that have curative effects, lower toxic side effects and lower cost of treatment. Objectives:The research is about the Herba Taxilli which parasitism in the Casuarina equisetifolia L. The Herba Taxilli is a parasitic plant, due to the different host plants, chemical constituents and pharmacological activities will change with the host to cure different diseases. According to the case observed, with the decoction of the single Herba Taxilli which parasitism in the Casuarina equiseti folia L, can reduce fever in children leukemia and realse the disease, but there is no relevant experimental studies reported. To research whether this particular species of Herba Taxilli is effective for the treatment of leukemia and further enrich the resources of Herba Taxilli, we get the medicine chemistry, analytical chemistry, erum pharmacology, and cell biology to combine together.We can preliminary select therapeutic active site of treating leukemic cells that provide a scientific basis to describe the material basis for the treatment of leukemiaMethods:Make a qualitative identification of chemical constituents of Herba Taxilli for the first time:using the powder microscopic identification and preparation of thin Layer chromatography, physical and chemical color reaction, to make a qualitative identification about the chemical constituents of Herba Taxilli.The experiment about serum containing Herba Taxilli:Make the decoction of Herba Taxilli, cytarabine, disinfection tablets compared with the control serum, all acting on the rabbit respectively, then separate serum containing drugs, using MTT colorimetric reaction and cell morphology to investigate the serum containing drugs'impact on the proliferation of leukemic cells K562 and HL60 for 24,48 and 72 h.The preliminary experiment about active site of Herba Taxilli:According to the chemical composition of pre-experimental results, using 95% ethanol and water as solvent, the ethanol extract is seperated by petroleum ether, chloroform, ethyl ether, ethyl acetate and n-butanol in turn to five fractions from small to large in polarity.Take the extracts role in leukemic cells K562 and HL60 that using MTT colorimetric reaction and cell morphology to investigate the effective anti-leukemia fractions of Herba Taxilli.The experiment about qualitative and quantitative identification of the active site:Through the the physical and chemical color reaction and UV spectrophotometer for qualitative and quantitative to active site of the Herba Taxilli. Results:①Powder microscopic identification experiment show that standard Herba Taxilli is similar with Herba Taxilli which parasitism in the Casuarina equisetifolia L. The wood fiber length and diameter only slightly different.②Qualitative identification of TLC show that standard Herba Taxilli and Herba Taxilli which parasitism in the Casuarina equisetifolia L. in the developing solvent:toluene:methyl formate:formic acid (15:13:3), observed at 365nm in the UV, it has the same color of the spots at the same position with the quercetin as reference substance. And the two Chinese herbal medicines have no significant difference in other spots when they are under the same treatment conditions.③Preliminary experiment of chemical composition results show that it may contain components such as amino acids, peptides, organic acids, phenolic compounds and tannins, sterols and triterpenoids, flavonoids, saponins, volatile oil and so on.④The serum containing test note:With the prolongation of the action time and the increase concentration of the drug, the positive control group Ara-C significantly inhibited both cell proliferation and the cell swell significantly; disinfection tablet group also inhibit both cell proliferation; the high dose group of Herba Taxilli have a weak inhibitory effect when they act on the two cells for 72h,it is worth further study.⑤The experiment about different extraction site of Herba Taxilli on the proliferation of leukemic cell K562 and HL60 suggest:with the prolongation of the action time and the increase concentration of the drug, ether,ethyl acetate and n-Butyl alcohol fractions from Herb Taxilli all have remarkably inhibited proliferation of the leukemic cell K562. When the reaction time is 72h, the half inhibitory concentration that ether,ethyl acetate and n-Butyl alcohol fractions from Herb Taxilli to inhibit proliferation of K562 cells are:(193.39±14.45)μg/ml, (83.23±2.79)μg/ml, (110.94±1.37)μg/ml; the ethyl acetate site has a strong inhibition of leukemic cell HL60, other parts of the inhibition was not obvious, when the reaction time is 72h, the half inhibitory concentration to HL60 cells proliferation is:(155.44±7.50)μg/ml. Morphological study results show that after 72h of drug action, the cell in the treatment group obvious shrinkage, the proliferation is inhibited, cell morphology is different, the nucleus are large and loose, the nucleolus decreases and there are a lot of cell debris.⑥The research about qualitative and quantitative of the active site shows that:ether,ethyl acetate and n-Butyl alcohol fractions from Herb Taxilli are the flavonoids. To determine total flavonoids in the standard Herba Taxilli and Herba Taxilli which parasitism in the Casuarina equisetifolia L by UV Spectrophotomety measured respectively 10.08mg/g and 17.05mg/g. The average recovery of total flavonoids measured respectively 97.92% and 99.81% with the relative standard deviation 2.56% and 3.07%. The total flavonoids content have slightly difference. The method is simple and rapid, its results are stable and reliable. So the method can be used for determination of total flavonoids of Herba Taxilli.Conclusion:The results show that the chemical properties of Herba Taxilli which parasitism in the Casuarina equiseti folia L and the standard Herba Taxilli have little difference, only the content are slightly different They can replace each other for use. The ether,ethyl acetate and n-Butyl alcohol fractions from Herb Taxilli all had remarkably anti-leukemia effective in vitro. They are the flavonoids. The research tip the flavonoids of Herba Taxilli have a certain effect on the treatment of leukemia. |
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