Screening The Hemostatic Active Fraction Of Artemisia Annua L. In Vitro And The Exploratory Development

Objective:Artemisin distilled from artemisia annua L. is the best medicine with the highest efficency, the most effective and the lowest toxicity in treating ague nowadays, and it is the only plant amedica which has been recognized to research and develope as the standards of western medicine research by the WHO. China is the largest country of origin of crude drug of arteannuin in the world, and it accounts 90% of the total production. At present, most studies focus on artemisinin and its derivatives, while the study and report about the other active components are rare. It has been reported in the literature that artemisiae annuae has remarkable pharmacological activities artemisia annua L.. Basing on the protection of the quality of artemisinin extraction, this paper purposed to further discover new indication of artemisia annua L. connecting with the record of traditional medicine. We explore other components of artemisia annua L. in order to utilize artemisia annua L. utmostly.The research screened the hemostatic active fraction of artemisia annua L. in vitro by pro-coagulant and hemostatic experiment. It can provide experimental data for the clinical application of artemisia annua which can be use as hemostatic. Essential oils from artemisia annua L. are major component besides the arteannuin. The essential oils own antimicrobial activities which have been reported in several literatures. Therefore, the topic plans to work out a novel haemostatic model with antibacterial and hemostasis through the mixture of the hemostatic active fraction and the essential oils. This topic has a certain academic value and potential prospects on the deep research of the artemisia annua resources.Method:1. Screening the evaluation of coagulation in vitro:①The crude extract of artemisia annua L.were lixiviated by 75% ethanol, and the extraction then were extracted in order by equal volume of petroleum ether, ethyl acetate, n-butanol. The clotting time (plate method for blood coagulation and test tube method) and plasma recalcification time were determined in vitro.②Screening the ideal evaluation method of coagulation, and verifying it through the traditional chinese herbal drug with hemostatic activity in the lab. 2. Determination of the active site:①Crude extract and the four extracts through organic solvent were tested by plate method for blood coagulation, test tube method and plasma recalcification time. The extract of n-butanol was purificated by polyamide, MCI, gel column in order.②Repeated comparative experiments of the coagulant effect of the part ofαby plasma recalcification time.③Analysis of the effective parts: The effective components were analyzed by chemical identification. It mainly contents tannins, flavonoids, alkaloids, organic acids, amino acids, volatile oil, protein, sugars, anthraquinones, and saponins.3. The research of the novel dosage form with antibacterial and hemostasis: first, determinating the antimicrobial and hemostasis of the mixture which were mixed byαand essential oils as a certain percentage. Next, we yield a more reasonable model through the design of the novel dosage form with antibacterial and hemostasis.Results:1. Screening the evaluation method of coagulation: The clotting time (plate method for blood coagulation and test tube method) and plasma recalcification time were detected in vitro. The crude extract of artemisia annua L. had shorter coagulation time. The comparison of the pro-coagulant effect of the extracts by the four organic solvent in vitro showed that artemisia annua L. extract by n-butanol had the shortest plasma recalcification timeand the strongest pro-coagulant effect in vitro. Plasma recalcification time is more sensitive and rapid for the clotting of plasma. Besides, the end of trial is clear and the results can be copied.2. Determination of the active site:①The comparison of the pro-coagulant effect of the extracts in vitro showed that the extract by n-butanol andαhave strong pro-coagulant effect.②The shorten rate of clotting time are followed by: The crude extract(8.51%); the extract by n-butanol(14.89%); the water part after Polyamide column(22.11%); the part ofα(27.37%). Sc, Sd and the remainder are significantly lower than the crude extract and even non-condensable. The coagulation time and shortened rate of coagulation time of the part ofαwere significantly shorter and higher than those before extraction, suggesting that the part ofαmay be the hemostatic effective parts of artemisia annua L..③Analysis of the effective parts: The effective components were analyzed by chemical identification. Qualitative analysis showed that the effective parts of artemisia annua L. were mainly tannins, alkaloids, organic acids, sugars, and anthraquinones.3. The research of the novel dosage form with antibacterial and hemostasis:①The mixture were mixed byαand essential oils as 1:1, and the results of the antimicrobial and hemostasis of it showed that effectives each other were not weaken, while a certain degree of contrast enhancement.②We yield a more reasonable model through the design of the novel dosage form with antibacterial and hemostasis.it is four layers patch: Back lining, pressure-sensitive adhesive PSA withα, essential oils layer and anti-stick layer.Conclution:The crude extract and the extract of n-butanol were purificated by polyamide, MCI, gel column in order. Determining the part ofαis the hemostatic active fraction of artemisia annua L.. The preliminary experiment through the oil mixed with the part ofαproved that it has feasibility of theoretical to exploit a novel hemostatic patch with antibacterial and hemostasis. Meanwhile, the model of four layers patch provides a clear direction. artemisinin is mainly reside in part of petroleum ether of artemisia annua L., and the essential oils in this subject can be obtained from the pre-treatment in extraction of artemisinin, while the part ofαcan be gained from the residue after extraction of artemisinin. Therefore, on the one hand, it can stop bleeding effectively, and prevent infection when bleeding. It also laies the foundation to look for the convenient and ideal traditional chinese herbal dressing for the hemostasis of the wound surfaces; On the other hand, the main materials of the model can be regard as the residual product of arteannuin, and it can expand the multiple use of artemisia annua L..