| Chuanxiong Rhizome,a traditional Chinese medicine,is derived from the dried rhizomes of Ligusticum chuanxiong Hort.,an umbelliferous plant.It is a commonly used drug for activating blood and dissolving stasis.In recent researches,the quality evaluation of Chuanxiong Rhizome is mainly measured by determining the contents of ferulic acid,ligustilide,Senkyunolide A and other active components.In Chinese Pharmacopoeia 2015 edition,the quality of Chuanxiong Rhizome was evaluated by using alcohol-soluble extracts(> 12%)and ferulic acid(≥ 0.1%)of Chuanxiong Rhizome as indicators.However,these two indicators are unstable and difficult to standardize.In this connection,in order to provide a basis for the quality evaluation of Chuanxiong Rhizome,the anticoagulant activity,anti-gel reactivity and anti-platelet aggregation activity of Chuanxiong Rhizome were quantitatively determined by using sodium ferulate as the positive drug.This research,is to develop a biological assessment to quantitative determining the efficacy of Chuanxiong Rhizome on activating blood and dissolving stasis.First,quantitative determination of anticoagulant activity of Chuanxiong Rhizome:(1)By investigating the factors influencing the quantitative determination of anticoagulant activity in vitro,and taking prolonging activated partial thromboplastin time(APTT)as an indicator,a quantitative method for the determination of anticoagulant activity of Chuanxiong Rhizome was developed to evaluate the quality of Chuanxiong Rhizome and Chinese patent medicines.That is,the rabbit blood was centrifuged at 3000 rpm for 10 minutes to obtain plasma.The store time of plasma was 3 hours.Sodium ferulate was used as the positive drug.And it showed a good linear relationship with APTT elongation rate in the range of 1 to 5 mg/m L(r = 0.9955,n = 5).After extraction of Chuanxiong Rhizome powder with ethanol for 3 times,the filtered drug residue was extracted with water for 3 times.The total extract of Chuanxiong Rhizome was used as the test solution.According to the reliability testing of amount reaction of parallel line analysis(2.2),(regressor P value < 0.01,deviation from parallel P > 0.05)in the Pharmacopoeia,the testing time should not exceed 3 times.The RSD value of the reproducibility experiment was 9.34%(n = 6),and the RSD value of the precision experiment was 9.59%(n = 6),the confidence limit was 11.15%(n = 6),less than 20%.These results showed this method is accurate and reproducible.(2)The amounts of anticoagulating bioactivity were significant difference among various types of Chuanxiong samples,i.e.5.431~7.620 U/g for 5 Chuanxiong raw materials,5.910 and 3.017 U/g for Chuanxiong decotion pieces and processed slice with yellow wine,14.516 and 29.035 U/g for Tongmai Granule and Xuefu Zhuyu Pill.The developed method can accurately quantify the level of anticoagulating bioactivity in Chuanxiong raw materials,decoction pieces and related Chinese patent medicines,and assess their quality.Second,quantitative determination of anti-gel reaction activity of Chuanxiong Rhizome:(1)By investigating the factors influencing the quantitative determination of anti-gel reaction in vitro,and taking the dose of fibrinogen in the thrombin-fibrinogen reaction as an indicator,a quantitative method for the determination of anti-gel reaction activity of Chuanxiong Rhizome was developed to evaluate the quality of Chuanxiong Rhizome and its Chinese patent medicines.That is,the Chuanxiong powder was extracted with ethanol three times,and the filtered drug residue was further extracted with water three times to obtain a total extract of Chuanxiong Rhizome.The working buffer was the mixture of saline and phosphate buffer(p H 7.4)with the ratio at 1:17.Then,the total extract of Chuanxiong Rhizome was prepared into a 40 mg/m L test solution by using working buffer.The bath time for thrombin-fibrinogen reaction was 10 min.According to the direct measurement method,the number of measurements was not less than 4 times.The RSD value of the reproducibility experiment was 4.00%(n = 6).The RSD value of the precision experiment was 7.82%(n = 6).And the confidence limit was 12.69%(n = 6),less than 20%.The results show that the method is accurate and reproducible.(2)Five Chuanxiong medicinal materials,2 Chuanxiong decoction slices(Chuanxiong decoction slices and processed liquor slices),and 2 Chinese patent medicines related to Chuanxiong Rhizome(Tongmai Granules and Xuefu Zhuyu Pills)were collected from different producing areas and medicinal materials markets,decoction companies and pharmacies.Their anti-gel reaction activities was determined.The anti-gel reaction activity of 5 Chuanxiong medicinal materials was 0.72~1.14 U/g,the Chuanxiong decoction pieces and processed liquor slices were 1.25 and 0.67 U/g.Tongmai Granules and Xuefu Zhuyu Pills were 2.516 and 2.56 U/g.The developed method can accurately quantify the level of anti-gel bioactivity in Chuanxiong raw materials,decoction pieces and related Chinese patent medicines,and assess their quality.Third,quantitative determination of anti-platelet aggregation activity of Chuanxiong Rhizome:(1)Based on the early laboratory researches of determining anti-platelet aggregation activity method of the water extract in Chuanxiong Rhizome,this research is to quantitative determine of anti-platelet aggregation activity of total extract of Chuanxiong Rhizome.After extraction of Chuanxiong Rhizome powder with ethanol for 3 times,the filtered drug residue was extracted with water for 3 times.The combination of alcohol extract and water extract was concentrated into about 60 m L in the reduced pressure,and transferred to a conical flask.The mixture was evenly mixed and weighed,that is,the total extract of Chuanxiong Rhizome.(2)Five samples of Chuanxiong medicinal materials,2 Chuanxiong decoction slices(Chuanxiong decoction slices and processed liquor slices),and 2 Chinese patent medicines related to Chuanxiong Rhizome(Lemai Granules and Quick-acting Jiuxin Pills)were collected from different producing areas and medicinal materials markets,decoction companies and pharmacies,and their anti-platelet aggregation activities was determined.The results show that the anti-platelet aggregation titer of 5 Chuanxiong medicinal materials were 0.56 ~ 0.75 U/g.Chuanxiong decoction pieces and processed liquor slices were 0.67 and 0.30 U/g.Tongmai Granules and Quick-acting Jiuxin Pills were 0.92 and 0.51 U/g.The developed method can accurately quantify the level of anti-platelet aggregation activitiy in Chuanxiong Rhizome medicinal materials,decoction pieces and related Chinese patent medicines,and assess their quality.Fourth,determination of anti-thrombotic effect of Chuanxiong Rhizome in vitro: used the traditional method of whole blood clot lysis,to determine of antithrombotic effect of Chuanxiong Rhizome in vitro.Low-dose Chuanxiong Rhizome extract solution does not have significance until the dose is increased to 25 mg/m L.The results showed that Chuanxiong Rhizome has a certain antithrombotic effect.Fifth,effect of Chuanxiong on improving microcirculatory disorder in mouse auricle:(1)After adding the adrenaline hydrochloride to the auricle of the mouse,the difference of micro-vascular diameter between normal group and model group was significant.Which indicating that the mouse auricle micro-circulatory disorder established was successfully modeled in this research.(2)The positive drug sodium ferulate and different concentrations of Chuanxiong Rhizome extract solution can significantly change the rate of changes in the diameter of auricular micro-vessels in mouse after 5 days of administration,indicating that both sodium ferulate and Chuanxiong Rhizome have a clear effect on improving the auricular micro-circulation disorders in mouse. |
PDF Full Text Request