| Striga (another names Dujiaogan, Ganjicao, Xiaomichong) is a kind dry herb of Striga asiatica (L.) O. Kuntze, from f igwort family. It is collected in summer and autumn, processed by washing clean and open-air drying.Striga of the family Scrophulariaceae is semiparasitic or parasitic on the roots of other plants. There are about 20 varietals spreaded in the tropics and subtropical zone of Asia, Africa, Australia. Striga asiatica, S. hermonthica, S. gesnerioides are the main harmful weeds in subtropical area.Striga is the traditional medicinal plants in folk in the South.It is usaully used to treat pedo-anorexy and dyspepsia, combined with Chinese hawthorn leaf, corium stomachium galli, byne, milllet sprout etc.There are seldom reports about the research of Striga.It is known as a folk medicinal herbs used for chilren' s malnutritional stagnation. No literature records on the research of the ingredients and the preparation. In this article ,research has done on the ingredients in Striga plants, and two typical chemical components have obstracted. All the basic research is prepared for the establishment of qualitative control method of Striga plants. Typical ingredients will help to optimize the extraction technology of Striga plants and the purification technology of extracted active components. Therefore, both finger prints and qualitative control methods of Striga plants and active components will be established at the same time. All the research and methods will be reported at length as follows.Part I Investigation of Literature reportsGreat many researches reports on Striga medical materia and have been read. After summarizing all the preceeding research fruits and existing problems, research content and objection are brougt out in this article, aiming to solve the problems about Striga medical materia and improve weak qualitative research.Part II Experimental ResearchSection One The separation and identification of the chemicalcomponents of StrigaChemical components of Striga are studied in this article for the first time. The powder of the herb was extracted by different methods, then obstracted by silica gel column chromatography, and two chemical components were gained. By their chemical reaction and spectral data of IR, MS, PMR, CMR, they were identited as pelargidenon ( I )and quercetin-3-o-rhamnoside(quercitrin) (II).Section Two Study on extraction technology of Striga total flavonoids 1. Study on ethanol backstreaming extracting technologyAccording to the research fundament and status, specific experimental situation, adopt ethanol backstreaming extracting methods. After inspecting the influence on the extracting results of each factor, ethanol concentration, extracting volume, extracting time, extracting frequency by simplex-factor test methods, determin the level of each factor, then optimize all the conditions of ethanol backstreaming extraction technology "?by orthogonal test. Resluts as follows:1.1 Inpection on different extracting ethanol concentrationInpect on and analyse the extracting resluts of diffrernt concentration of 50%,€0%,70%,80%, 95%. Consider different extract yields of Striga total flavonoids and their content of total extract, choose 70% ethanol as the solvent.1.2 Inspection on different extracting timeInspect on and analyse the resluts of diffrernt extracting time during for for2.0hr, 1.5hr, l.Ohr, 0. 5hr separately, consider respective extracting results,then choose extracting time during for lhr as the optimal condition. 1. 3 Inspection on different amount of extracting solventInspect on and analyse the resluts of diffrernt quantity of extracting solvent of 14, 12,10, 8, 6 times volume of the medicinal powder separately and extracting for 1,2,3 times respectively. Consider different extract yields of Striga total f lavonoids and their content of each extract choose the amount of solvent of 10 times volume of the medicinal powder and extracting for 3 times as the optimal condition. 1. 4 Optimizing Striga backstreaming extracting technology by orthogonal testCombine all the above-mentioned technic parameters' extracting results and pratical producing conditions. Choose the solvent quantity, backstreaming extracting time, extracting frequency as the considerated factors and set three levels. According to the factor-level table .employ orthogonal table to assey all the factors. Adopt ultraviolet absorption method to determin the Striga total flavonoids in the extract, indicated by the content of pelargidenon. The optimal conditions of Striga ethanol backstreaming extraction technology are :70% ethanol of 6 times volume of medicinal materia powder, backstreaming extracting for 3 times,30minutes per time.2 Study on ethanol ultrasonical extraction technologyOn the research base of ethanol backstreaming extraction technology, adopt ethanol ultrasonical extraction technology. After inspecting the influence on the results of each factor:ethanol quantity, ultrasonical extracting time, extracting frequency, basicly establish Striga ethanol ultrasonical extraction technology. The optimal conditions of Striga ethanol ultrasonical extraction technology are :70% ethanol of 20 times volume of medicinal materia powder, ultrasonically extracting for 2 times,2. 5hrs per time.Section Three Study on the Striga total flavonoids depurationtechnology1. Bolting Striga total flavonoids macroporous-adsorptive-resins- depuration technologyBy comparing different depuration methods, optimize the Striga' extract purification technology. Inspect resin type, dynamic and static saturate adsorptive quantity and eluting rate, passing-through column rate, ethanol eluding, concentration and volume. Determin macroporous adso'rptive resin depurationtechnology.Results as follow:1.1 Inspection on various different static absorptive quantity of Striga total flanonoids in different types of resinsDraw free water from the dealt resins of different types, LSA-21 resin^ LSA-40 resinN F resin, SLA-5B resin, FL-1 resin, FL-2 resin, XDA-16B resin, D101 resin, and accurately weigh certain amount and put them into a conical flask respetively. add cerntain extract accurately to each conical flask, shake for 2 hrs, 30 seconds per time, with internal of 10 minutes. Then stand for 24hrs and make the extract absorped thoroughly, and essay on the static absorptive.1.2 Study on the various static eluting efficiency of Striga total flanonoids in different type of resinsFilter off all the thoroughly-absorping resins respectively, dry the samples by filter papers,then put into the conical flask separately and add certain amount of 70% ethanol,shake for 2hrs, 30 seconds per time, with internal of lOminutes.At last stand for 24hrs and prepare for the inspection on the static eluting quantity.By the static obsorption and desorption tests, optimize the AB-8 macroporous adsorptive resin as column chromatography resin(static absorption quantity is 27. 69 + 0.198mg sample per lg resin, static desorption ratio is 94. 68±0. 231%). 1. 3 Study on AB-8 aacroporous adsorptive resin dynaaic adsorption quantityDraw the free water from the dealt AB—8 resin and put into chromatographic column. Accurately suction certain amount of extract and add it to the chromatographic column. Elute the colume with solvent, at the rate of 2 times volume of the column volume per hour. Collect eluant, dissolve to certain volume for dynamic absorption quantity. Result: the dynamic absorption quantity of AB—8 resin is 25.65 +0. 19mg sample per lg resin.1.4 Study the influence of the adsorped sample of different concentration on asborption efficencyDraw the free water from the dealt AB—8 resin,then prepare 6 certain amount of resins and put them into 6 chromatographic columns separatively. Accurately suck 6 adsorped samples of different concentration and add them to the 6 chromatographic columns respectively. Elute the colume with solvent, at the rate of 2 times -volume of the column volume per hour. Collect eluant, dissolve to certain volume for the study of the influence of the adsorped samples of different concentration on asborption efficency. Result:the optimal concentration of adsorped sample arrangs from 1. 1 to 1. 6mg materia per ml.1. 5 Study on the influence of eluting rate on absorption efficencyDraw the free water from the dealt AB—8 resin, then prepare 6 piles resin of certain amount and put them into 6 chromatographic columns separatively. Accurately suck 6 adsorped samples and add them to the 6 chromatographic columns respectively. Elute the colume with solvent,at the separative rates of 1,2,3,4,5 times volume of the column volume per hour. Collect eluant respectively, dissolve to certain volume for the study on the influence of different eluting rate on asborption eff icency. Result:the optimal eluting rate is eluting 2-times-column-volume solvent in hour. 1.6 Study the influence of different solvents on the absorption efficencyDraw the free water from the dealt AB—8 resin, then prepare 6 piles resin of certain amount and put them into 6 chromatographic columns separatively. Accurately suction 6 adsorped samples and add them to the 6 chromatographic columns respectively. Elute the colume respectively with different solvents:200ml distilled water, 100 ml 20%Et0H, 100 ml 50%EtOH, 100 ml 70%EtOH, 100 ml 95%EtOH in order, at the rate of eluting volume of 2 times column volumes per hour. Collect eluant step-by-step, dissolve to certain volume for the study on the influence of different solvents on the absorption eff icency. Result:elute the water—solubility dopant with distilled water firstly, then elute Phenols with 20%EtOH, finally elute with 70 %EtOH and collect the last eluant. 1. 7 Study the influence of different volume solvents on the absorption efficencyDraw the free water from the dealt AB—8 resin, then prepare 6 piles resin of certain amount and put them into 6 chromatographic columns separatively. Accurately suction 6 adsorped samples and add them to the 6 chromatographic columns respectively. Elute the colume individuatelly with different solvents:200ml distilled water, 250ml distilled water, 300ml distilled water, 200 ml 20%Et0H, 300 ml 70%EtOH in order, at the rate of eluting volume of 2 times column volumes per hour. Collect eluant step-by-step, dissolve to certain volume for the study on the influence of different volume solvents on the absorption eff icency. Result: elute the water—solubility dopant with 200ml distilled water firstly, then elute Phenols with 200ml 20%Et0H, finally elute with 200ml 70%Et0H and collect the last eluant. 1.8 Study on the AB—8 resin ' reproducibility and purificationThe tests of optimizing every technic parameters indicate that, the adsorped sample' concentration shuold arrang from 1. 1 to 1. 6mg materia per lml, elute w^th200ml distilled water, 200ml 20%Et0H, 200ml 70%Et0H in order, at the rate of eluting /volume of 2 times column volumes per hour, collect the 70% EtOH eluant, then can gainthe total flavomoids of 63.12+0. 28%. sample, with good reproducibility (RSD=O. 45%) and the purification of 245.89%.2. Study on the optimization of polyamide depuration technology of Striga totalflavonoidsOn the research base of macroporous adsorptive resins-depuration technology,optimize Striga' extract polyamide depuration technology, and inspect the the dynaminc and static saturate adsorptive quantity, eluting rate, passing-through-column rate,ethanol eluting concentration and eluting quantity. Establish polyamide depuration technology' optimal conditions.The datas from the static absorpotion and static desorption experiment arrive at that the static adsorpting quality is 46. 31+0. 57mg extract per lg polyamide and the static desorption rate is 93. 37±0.-64%. Technic parameter optimizing experiments indicate that eluting volume of 2 times column volumes per hour per hour, eluting with 200ml distilled water, 200ml 20%Et0H, 250ml 95%EtOH in order, and assembling the 95% EtOH eluant are the optimal parameters, finally can gain the total flavomoids of 63. 12 ±0.28% sample, with good reproducibility ( RSD = 0. 40 % ) and the wonderful purification of 254.54%.Section Four Study on the Striga finger print 1.Methods of HPLC finger printEmploy the optimal technic parameters to extract Striga medical materia. Dissolve the extract to certain volume and filter the solution with 0. 45Mm micropore film. Pour the filtrate into the specific bottle. Inspect all the possible factors influencing on the HPLC finger print.Results as follow: 1.1 Inspection on mobile phase gradientFilter the Striga extract with 0. 45Hm micropore film, then pour the filtrate into the specific sample bottles, place the botttles in the specimen disc. Methods: Performed on the luna column,with detective wavelength at 340nm and flow rate at 0.8ml/min and column temperature at 25 °C ,setting different mobile phase gradients, then analysing the best eluting gradient. Results:mobile phase is composed by methaol(B)-0. 5% phosphonic acid(D) with four eluting gradients. The composing ratio changs with time flowing, from initial ratio of 35%B:6B%D at 0 minute, after 3minutes the ratio is at 35%B:65%D, after another 9minutes, the ratio is 45%B:55%D, atthe 38th minute, the ratio changs at 62%B:38%D, at the 45th minute the ratio is 66%B:34%D, finally the ratio changs back to the initial one 35%B:65%D at the 60th . 1. 2 Inspection on different types of HPLC columnsTest different types HPLC columns' performance by following the optimal eluting gradient. Results:the performance of Dikma ( DiamonsilTMC18 250X4. 6mm ) column is the best.1. 3 Inspection on the influence on the elution efficency of different types of acid and their various concentration.Basing on optimal eluting gradient and separating column conditions, inspect different acids and acids of various concentrations' influence on the separating spectrumo After combining all the factors, determin methanol-2. 0% formic acid to be the elution system.1.4 Inspection on the influence on the elution eff icency of different eluting rates Keeping all the parameters unchanged, vary the flow rate of the mobile phase, theninspect different flow rate' influence on the separation of the chromatographic peaks, and determin the optimal flow rate condition. Results:the optimal flow rate is 0.8 ml flow pre minute.1.5 Inspection on the influence on the elution efficency of different column temperaturesKeeping all the parameters unchanged, vary the column temperature, then inspect different column temperature' influence on the separation of the chromatographic peaks, and determin the optimal column temperature condition. Results:the optimal column temperature is 30 °C1. 6 Determining all parameters for the finger printAfter optimizing all the parameters,the spectrum conditions of Striga total flavonoids.' HPLC finger print are as follows: Dikma ( DiamonsilTMC18 250X4. 6mm ) column as the performencing column wih four eluting gradients, methanol-2%formic acid as the mobile phase, flow rate at 0.8ml flow per minute, column temperature at 30°C,and the detective wavelength at 340nm.2. Study on the finger prints of both Striga medical materia and the purified productBasing on several main points such as reproducibility, stability, precision an* so on, all the test results of the finger prints of both Striga medical materia^ndthe purified product accord with the requirement.3. Analysis and evaluation on the HPLC finger prints of both Striga medical materiaand the purified productAccording to all chromatographic conditions, characteristic absorption bands in the HPLC finger prints of both Striga medical materia and the purified product are separated clearly. The general peak features are nearly identital. This indicate that the components in different samples are almost the same. Resemblance evaluation conducted by computer also shows that there is a high coincidence between Striga medical materia and their purified product.Section Five Study on the quality criteria of Striga medical materia1. Determinination moisture contentAccording to the method of moisture content determination of Pharmacopoeia of the People's Republic of China (first edition in 2005, appendix K K ), total moisture content of Striga asiatica(L. )0. Kuntze medical material is not far from each other, in the range of 9. 52%""9. 97%. So it is possible to determin that the moisture content ratio shuold be under 11%.2. Determination of total ash and acid-insoluble ashAccording to the method of ash determination of Pharmacopoeia of the People's Republic of China (first edition in 2005, appendix DC K ), total ash content of Striga asiatica(L.)0. Kuntze medical material is in the range of 22. 6%~23. 4%, the content of acid-insoluble ash is in the range of 8. 7% 9. 1%.3. Determination of extractumAccording to the method of extractum of Pharmacopoeia of the People' s Republic of China (first edition in 2005, appendix X A), the content of water soluble extract is in the range of 14. 3% to 20. 7%, content of alcohol-soluble extractive is between 12. 0 % and 15. 4%.4. Residual volume of heavy metalsThe content of As, Pb, , Cd, Hg , Cu is determined according to As, Pb, Cd, Hg, Cu assay (atomic absorption spectrophotometry) of Pharmacopoeia of the People' s Republic of China (first edition in 2005, appendix IX B );The colour comparison between examined sample and standard Pb solution , the total amount of heavy metals (calculating with Pb) is carried out by check method of heavy metals in Pharmacopoeia of the People' s Republic of China (first edition in 2005;appendix IX D);the resultof atomic absorption spectrophotometry is following: lead(Pb) is 0. 79~0. 95mg/kg;chromium (Cd) is 0. 15mg/kg;copper(Cu)is 10mg/kg;arsenicl (As) is less than 0. 5mg/kg;mercury (Hg) is less than 0. lmg/kg. The result of heavy metals checking is following: total amount of heavy metals is less than lOppm;All these are in the permission range of ((green color trade standard about plant export and import of amedica , praeparatum)) , which is published by foreign trade economic cooperation department of the People's Republic of China .5. Pesticide residue of organochlorineAccording to pesticide residue assay of the People' s Republic of China (first edition in 2005, appendix K Q), pesticide residue of organochlorine is assayed by the gas chromatogram external reference method. The concentration of Chlorophenothane (DDT) is less than 1. OX 10-3mg/kg j benhexachlor (BHC)is 1.2X10-3mg/kg: quintozene ( PCNB ) is 7.5X 10-3mg/kg;Aldrin is less than 1. OX 10-3mg/kg. All these are in the permission range of ((green color trade standard about plant export and import of amedica , praeparatum)) , which is published by Foreign Trade Economic Cooperation Department of the People' s Republic of China .6. Content assayThe content of pelargidenon in Striga and their purified product (total flavonoids) of Striga asiatica(L. )0. Kuntze is determined by HPLC, the content of pelargidenon in medical material should not be less than 4. Omg/g, that means it should not be less than 0. 4%, the content of pelargidenon in total flavonoids should not be less than 70mg/g, hat means it should be not less than 7.0%...
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