Applied Research On Blood-activating Effective Parts Of Shaanxi Produced Yellow Oil Point Grass And Its Serum Fingerprint

Purposes:To discover material basis for the blood-activating function of Tricyrtis maculate(D.Don)Machride in Shaanxi province;to determine minimum effective dosage through comparing the serological fingerprints at different dosages;to choose the dosage form through comparing the serological fingerprints of the drop pills and tablets at the same dosage and to explore new ideas for the use of the serological fingerprints to guide the preparation design.Methods:such ways as traits,microscopy and thin layer chromatography are borrowed to establish the basis for pharmacognosy identification so that the herbal source may be determined;the system solvent method is applied to extract samples,and the reaction of the color inspection is used to identify the types of herbal ingredients.Under the coagulated-cold static-blood animal model,the mouse ear microcirculation tests at such different doses as 2.5g/kg,5g/kg and lOg/kg are to Preliminarily determine the dose,which contributes to the mouse ear microcirculation test,the bleeding or coagulation test,the rat anti-thrombosis test,and also compares the blood-activating roles of the extracted parts under the various solvents such as water,70%ethanol,methanol,ethyl acetate,butanol,95%ethanol.The thin-layer chromatography,with the containing serum(plasma)as the control,is to judge whether the spots are the same in color by the observation of the washed impurity and eluted parts under different concentrations of ethanol such as 20%,40%,60%,80%,and 95%,referring to the same Rf value of the control,so that the parts are merged;the mouse microcirculation test,the bleeding and coagulation test,the rat anti-thrombosis test,the rat acute ischemic cerebrovascular obstruction of different merged parts are to determine the effective blood-activating parts;The reaction of the color inspection is to determine the composition of the active site;Based on UV spectorophotometry and total flavonoids as an indicator,parallel experiments is to determine the extraction method,the amount of water and the number of boiling,the orthogonal test is to select parts of the extraction process of total flavonoids,and its validity is to verify;The AB-8,D-101,HPD-450,HPD-600,HPD-700 five species of macroporous resin are selected according to the amount of transferred total flavonoids from per gram purified resin as an indicator and observation on the process from the dynamic adsorption to desorption,and from the static adsorption to desorption.Then parallel tests are conducted to examine the effect of extracted liquid mass concentration,the ratio between diameter and height,the pH value,the sample rate,gradient elution concentration and etc.on the resin-purified process,and then to select the optimal condition of the purification and verify its validity;UV spectrophotometry is to determine the total flavonoid of the merged parts;high-rate liquid chromatography is to determine the Nicotiflorin content;and finally the fingerprints of total flavonoids are studied;HPLC method is used to select the blank serum-free peak with greater areas as the characteristic peak,and whose number and area are normalized by the formula(Xi,j for the total area at different times,j for the average value,Sj for the standard deviation,namely,X’i,j for the normalized value).Thus a consoled evaluation Yλ value is got from Yλ=(YA+Yn)/2(Yλ refers to the eigenvalue when wavelength is λnm;YA refers to the standardized value of the total area;Yn refers to the standardized value of the peak number).The biggest Y value is viewed as the best.After the related check of per Y value,combined with the RSD value of the reserved time,the fingerprints’similarity is described.With the help of the determination of serum fingerprint,which is established through High-rate liquid chromatography,the serum fingerprints of 0.08g/kg 0.17g/kg,0.5g/kg,0.75g/kg 1g/kg in 15min,45min,1.5h are conducted by comparing the serum fingerprints of 0.25g/kg,1.25 g/kg,2.5g/kg,5 g/kg,7.5g/kg,10 g/kg in 15min,30min,45min,1.0h,1.5h,2.0h,3.0h,4.0h,6.0 h,8.0 h;then based on differences between the two groups and combined with the standardized value,the minimum effective dose is to determine;Tablets and drop pills are made from the effective parts,and samples are tentatively determined by the preliminary examination of pharmaceutics quality and dissolubility study.Studying the serum fingerprints of tablets and drop pills of the same dose at 15min,30min,45min,1.0h,1.5h,2.0h,3.0h,4.0h,6.0h and 8.0h,according to their similarities and differences,and combined with the standardized value,the dosage form is determined.Results:Tricyrtis macropoda,produced in Qinling Mountains,is the dried whole plant of liliaceous Tricyrtis maculata(D.Don)Machride.Tricyrtis maculata(D.Don)Machride in Shaanxi contains phenolic compounds,free alkaloid,flavonoids,amino acids,polypeptide,protein,A-phenolic hydroxyl group or dihydroxyanthraquinone of the O-phenolic hyd roxyl structure,cyanide,polysaccharide,lactone coumarin class,etc.;and it excludes saponins,cardiac glycosides,coumarins,steroidal terpenes,essential oils,etc..extracted parts by water,by 70%ethanol,and by methanol all have remarkable function on improving blood circulation,namely,the extracted parts by water with the role in prolonging the mouse blood coagulation time,and extracted parts by water with the anti-thrombosis effect.The common components of the above three parts are the type of phenolic compounds,flavonoids,amino acids and peptide and protein,organic acids,anthraquinones and carbohydrates,and other ingredients.Parts eluted by 40%,60%,80%ethanol have the same color spots as the administrated animal blood at the same Rf value.The three are merged as one,the rest after merger as the other.The former can not only improve the mouse microcirculation and prolong the clotting time,but also have antithrombotic effect and significantly improve the rat neural behaviors caused by acute cerebral ischemic cerebral infarction,reducing the rat cerebral infarction area.Parts eluted by ethanol involve the total flavonoids,while the other contain phenolic compounds,amino acids and peptides,proteins,organic acids,anthraquinones,sugar and so on.The optimum extraction of total flavonoids follows:first soak for 15 min,then decoct within 40,35and 35 times water respectively forlh,30 min and 30 min.Of the five kinds of macroporous resin such as AB-8,D-101,HPD-450,HPD-600 and HPD-700,the AB-8 macroporous resin plays a prominent role in the purification of the medicinal total flavonoids.The best purification process parameter for the liquid concentration is 1.2g/ml;raw herbs:resin is 1:3;pH is 6.47;the ratio of diameter to height is 1 to 5;eluent concentration is 40%;sample rate is 1BV/h;elution speed is 1BV/h;elution volume is 2BV.After water extraction and alcohol precipitation,3000g medicine is purified into 281.8g from Parts eluted by 40%alcohol,88.3g from Parts eluted by 60%alcohol,and 48.2g from Parts eluted by 80%alcohol.In each dry extract,the total flavonoids content is 54.74%,36.57%,21.00%respectively.The total content of combined parts is 36.495%.Chromatographic column:Thermo Hypersil Gold;mobile phase:gradient elution between(A)C213N and(B)0.4%H3PO4-H2O:0 to 30min,A(%)15 to 25;30 to 80min,A(%)25 to 55;80 to 90min,A(%)55 to 95;flow rate:1ml per min;sample amount:10ul;column temperature:20℃;detection wavelength:360nm.Under the condition,the fireworkucosinolate content is 0.747%in herb and 1.075%in total flavonoid part,whose fingerprints show significant relation and great similarity.Chromatographic column:Thermo Hypersil Gold;mobile phase:gradient elution between(A)C2H3N and(B)0.2%H3PO4-H2O:0 to 30min,A(%)30 to 33;30 to 35min,A(%)33 to 38;35 to 40min,A(%)38 to 52;40 to 85min,A(%)52 to 70;sample amount:20ul;column temperature:20℃;detection wavelength:220nm.Under the condition,under such condition,the serum fingerprint method is tested as reasonable and available.There is no significant difference between groups such as 0.25g/kg,1.25 g/kg,2.5g/kg,5 g/kg,7.5g/kg,10 g/kg,0.08g/kg,0.17g/kg,0.5g/kg,0.75g/kg and 1g/kg,whose Ymax is respectively 1.803,1.513,1.429,1.392,1.313,0.703,0.633,0.243,1.146 and 0.861,and among which the Y value of 0.25g/kg is the greatest,so 0.25g/kg is selected as the lowest effective dose.There is no significant difference between tablets and drop pills.The tablets Ymax is 1.846 in 30min,and the drop pills Ymax is 1.027 in 2h,so it’s better to choose the tablet as research agents to develop formulations.Conclusion:The established identification base of raw pharmacognosy,HPLC used to determine effective part fingerprints and serum fingerprints can be found to be reasonable and available.Total flavonoid is the basic substance for blood-activating function of Tricyrtis maculate(D.Don)Machride in Shaanxi province.0.25g/kg is the most proper dose,and tablet is better than drop pill.It’s available to guide the formulations selection,lying firm foundation for developing form to practical research,development and utilization of this herb resource.