A Preliminary Study On Chemical Constituents Of Bauhinia Championii Benth And Anticoagulation Effect Of Total Flavonoids Of Bauhinia Championii Benth

Purposes: Bauhinia championii Benth is the dry rattan bark of Bauhinia championii(benth.)Benth, being a featured drug for arthralgia treatment. It is mildnatured, bitter and non-toxic with the efficacy of dispelling wind and eliminating dampness, and it is clinically used for the treatment of rheumatoid arthritis, lumbago, bruises embolism, etc. Modern pharmacological studies show that the extract of Bauhinia championii(benth.)Benth mainly contains flavonoids, polysaccharide and volatile oil, with a strong anti-inflammatory analgesic, anti-rheumatism, and antiplatelet aggregation activity. The present study focuses on the chemical components of Bauhinia championii(benth.)Benth, the anticoagulant efficacy of total flavonoids of this plant and the method for the content determination of effective components of it, which lays a foundation for the study and explanation of anticoagulant mechanism of action of Bauhinia championii(benth.)Benth’s total flavonoids and the development of new anticoagulants.Methods:(1) The researcher studies the chemical components in the ethyl acetate part of Bauhinia championii(benth.)Benth. The technology of silica gel column choromatography and gel column choromatography is applied for the separation and purification of its chemical components.(2) The method of RP-HPLC is employed to measure the content of 5,7,3’,4’,5’-pentamethoxyflavone and 5,6,7,3’,4’,5’-hexamethoxyflavone in the total flavonoids of Bauhinia championii(benth.)Benth.(3) A Thin Layer Choromatography(TLC) for the two kinds of methoxyflavones in the total flavonoids of Bauhinia championii(benth.)Benth is established in the present study.(4) The researcher does research on the anti-coagulation effect of total flavonoids of Bauhinia championii(benth.)Benth. The effect of total flavonoids of the plant on the mice in vivo bleeding time and clotting time is observed with cutting tail procedure and glass capillary procedure. Tude method is applied to determine the effect on the mice in vitro clotting time.Results:(1) By repeated silica gel column choromatography and gel column choromatography, two compounds can be extracted in the ethyl acetate of Bauhinia championii(benth.)Benth. They can be identified as 5,7,3’,4’,5’-pentamethoxyflavone and 5,6,7,3’,4’,5’-hexamethoxyflavone by the ways of physical and chemical properties identification and modern spectroscopy combing with document control.(2)5,7,3’,4’,5’-pentamethoxyflavone and 5,6,7,3’,4’,5’-hexamethoxyflavone showed a good linear relationship with the peak area respectively in 1.974~13.818μg·ml-1(R2=0.9999)and 2.563~17.938μg·ml-1(R2=0.9998). The average recovery rate is 100.65% and 99.55% respectively and RSD is 1.89% and 0.91%. The mass fraction of 5,7,3’,4’,5’-pentamethoxyflavone and 5,6,7,3’,4’,5’-hexamethoxyflavone in total flavonoids of Bauhinia championii(benth.)Benth is respectively 42.00% and 35.37%.(3) When 5,7,3’,4’,5’-pentamethoxyflavone and 5,6,7,3’,4’,5’-hexamethoxyflavone are made to be the reference substance, and petroleum ether and ethyl acetate(3: 10) to be the development system, the thin-layer identification of total flavonoids of Bauhinia championii(benth.)Benth is of distinctive feature and strong specificity.(4)(1) The effect on mice in vivo clotting time(glass capillary method): all of the three dose groups of total flavonoids of Bauhinia championii(benth.)Benth can extend the clotting time in mice, moreover, both middle-dose group and high-dose group compared with the blank group can significantly prolong the clotting time.(2) The effect on mice in vivo clotting time(cutting tail method): the bleeding time of those three dose groups is obviously longer than that of the blank group.(3) The effect on the plasma recalcification time in mice: those three dose groups can dramatically lengthen the plasma recalcification time.(4) The effect on mice in vitro clotting time: middle-dose group and high-dose group, which have been compared with the blank group, can significantly prolong the in vitro clotting time in mice.Conclusions:(1) In the ethyl acetate part of Bauhinia championii(benth.)Benth, two chemical compounds are isolated.(2) A method of using HPLC to measure two kinds of methoxyflavones in the total flavonoids of Bauhinia championii(benth.)Benth is established. This method can be easily and accurately operated, with good separating effect, precision, reproducibility and stability. It can be applied to measure the mass fraction of 5,7,3’,4’,5’-pentamethoxyflavone and 5,6,7,3’,4’,5’-hexamethoxyflavone in total flavonoids of Bauhinia championii(benth.)Benth.(3) The total flavonoids separated from the ethyl acetate part of Bauhinia championii(benth.)Benth have significant anti-coagulation activity, and thereby have a good application prospect.