Probe About The Pharmacy Part Of Gengnian Granules

Objective: On the formal basis of the technology and quality controlling studying of Gengnian granules, further studying and improving the technology and further study quality controlling of Gengnian granules. Applying new process and new 'technology as SFE-CO2, high molecule wrapping technology, studying extract and protect effective component of compound TCM; studying the relation between technology and components extracting, comparing different technologies to medicinal materials extracting; making a thorough studying on high molecule wrapping extraction of compound TCM; applying several verify methods to identify molecule wrapping, and investigate its stability, calculate its effective time. On the basis of the modem fingerprint atlas quality controlling technology, for the characteristic of TCM quality controlling, put forward setting up "complete fingerprint atlas system" to control the TCM quality, initially probe to formulate the "complete fingerprint atlas system" of Gengnian granules.In the direct of TCM compatibility theory of "Jun, Chen, Zuo, Shi", utilizing the studying out come modem phytochemistry, computer technology, applying advanced analysis diplomacy, completely improve the technology and quality of Chinese drugs preparation, to establish the basis of modem Chinese drugs preparation.Method: (1) Applying HPLC determine Ferulic acid and a-cyperone contents of extraction of angelica, atractylodes macrocephala, nutgrass galingale rhizome, amomi of Gengnian granules (call "A"), and review methodology. The content determine method of Ferulic acid: Aginent 1100 HPLC instrument, C18 column (Kromasil 250×4.6mm, 100-5), mobile phase acetonitrile-0.085% phosphoric acid(17: 83), detect wavelength 316nm, theoretical plate number no lower than 5000 refer to Ferulic acid peak, sample size 5μl; The content determine method of a-cyperone: Aginent 1100 HPLC instrument, C18 column (Kromasil 250×4.6mm, 100-5), mobile phase methanol-water(80: 20), detect wavelength 254nm, theoretical plate number no lower than 1500 refer to a-cyperone peak, sample size 10μl.(2) Deploying mono-factor, taking the extraction as Ferulic acid and a-cyperone as the reviewing index, review the effect of medical materials grinding size, the variety of entrainer, the dosage of entrainer, extracting pressure, resolution pressure, CO₂ flow rate, extracting temperature, extracting time to the SFE-CO₂ extracting; and further, and take uniform design to optimize the technology condition of the main factors as extracting pressure, extracting temperature, extracting time, resolution pressure of SFE of A.Taking the extracting quantity as the review index, chose the method of steam distillation of A whether extract divide or together. The main factor affecting the method of steam distillation is imbitition quantity, soaking time, steaming time, thus taking the volatile oil extracting quantity and the extracting quantity of a-cyperone as the review index, applying orthogonal experiment optimized the imbitition quantity, soaking time, and steaming time.Taking the extracting quantity of Ferulic acid and a-cyperone as the review index, comparative studied the two kinds technology of SFE-CO₂ technology and steam distillation technology of A, reviewed the effect of the two kinds technology to extracting quantity.Taking the extracting quantity of Ferulic acid as the review index, probed the relation between the extracting component variety of angelica and the SFE-CO₂ technology parameter. Taking the extracting quantity of Ferulic acid as the review index, compared the effect of different technology as SFE-CO₂ technology, steam distillation technology, aqua extracting after steam distillation technology, ethanol percolating technology, ethanol backstreaming extracting technology of angelica to the extracting quantity of Ferulic acid.(3) Taking the clathrate availability as the review index, applying single factor experiment, reviewed the effect of clathrate variety, clathrating method, clathrate dosage, clathrating temperature, clathrating time to the extracting clathrate availability of A. Taking the clathrate availability as the review index, applying orthogonal experiment to optimize the three technology parameter asβ-CD dosage, clathrating temperature, stirring time.Carried out qualitative test to the extractionβ-CD clathrate of A: carried out lamellar detect toβ-CD clathrate, taking silica G thin layer plate, taking ligarineethyl acetate (17: 3) as developer, putting at uviol lamp(365nm) to postmortem; applying UV method, taking the location and hight of absorbing curve and absorbing peak to judge the forming information of the clathrate, take the UV-2401 UV instrument of Japan DJ, scanned the 13 -CD clathrate in 200～400nmCarried out stability test to the extractionβ-CD clathrate of A: detected the equilibrium moister absorption rate ofβ-CD clathrate; applying chemical kinetics method——temperature coefficient method (Q10 method)to anticipate the expiration date ofβ-CD clathrate.(4) Applying HPLC method, probed to formulate "the complete fingerprint system" of Gengnian granules, probed to formulate fingerprint of glycoside of Gengnian granules. Taking peoniflorin as the reference, Aginent 1100 HPLC instrument, C18chromatographic column (Kromasil 250×4.6mm, 100-5), Aginent data handing software, taking acetonitrile-0.1% H₃PO₄ aqueous solution (80: 20) as mobile phase to gradient elution, flow rate 1.00ml/min, column temperature room temperature, detect wavelength 225nm, sample size 10μl, record time 50min, taking the retention time and peak area of peoniflorin chromatographic peak (S peak) as 1 to calculate relative retention time and relative peak area ratios of other peak; also review methodology; and applying " the Chinese medical materials chromatograph fingerprint similarity appreciation system" of nation pharmacopoeia board to appreciate similarity.Prepared medical materials experiment solution and vacancy medical materials experiment solution of Gengnian granules, referring to the formulating method of formulating fingerprint of glycoside of Gengnian granules, formulated each fingerprint, and carried out to contrast analysis with the fingerprint of glycoside of Gengnian granules.Results: (1) The results of reviewing methodology of Ferulic acid: there was well linear correlation in the range of 0.050～0.800μg of Ferulic acid, regressionequation: Y=4130.8x+4.9042, R²=0.9999; precision experiment showed that the precision is well, RSD0.62%; stability experiment showed that the peak area of experiment solution is stable in 12 hour, RSD1.25%; reproducibility experiment showed the reproducibility is well, RSD3.18%; sample spotting recovery experiment showed it correspond with the regulation, the average recovery is 96.50%. The results of reviewing methodology of a-cyperone: there was well linear correlation in the range of 9092～79.36μg of a-cyperone, regression equation: Y=273.34X+26.116, R²=0.9999; precision experiment showed that the precision is well, RSD1.42%; stability experiment showed that the peak area of experiment solution is stable in 12hour, RSD1.09%; reproducibility experiment showed the reproducibility is well, RSD0.76%; sample spotting recovery experiment showed it correspond with the regulation, the average recovery is 98.47%.(2) The results of the single factor experiment method of SFE-CO₂ method of A: the grinding size of medical materials can affect little to the extraction quantity of Ferulic acid and a-cyperone, for extracting the active components as far as possible, adopt the coarse grain would be well; the variety of entrainer can affect obviously to the extraction quantity, and the extraction quantity of 95% ethanol and methanol is the max, taking care of the safety and economy of the extracting operation, adopt 95% ethanol would be well; when the dosage of entrainer reach 20% of mecical materials, the extraction quantity keep stability, taking care of the economy, adopt 20% would be well; the more the pressure of extracting, the more the pressure of resolution, the more the CO₂ flow rate, the more the extraction quantity, when thepressure of extracting reach 25～30MPa, the pressure of resolution reach 8～10MPa, the CO₂ flow rate reach 10～12L/h, the extraction quantity can keep stablity, taking care of the safety and economy of the extracting operation, the pressure of extracting adopt 25～30MPa, the pressure of resolution adopt 8～10MPa, the CO₂ flow rate adopt 10～12L/h; when the extracting temperature reach 40～45℃, the extraction quantity keep stability, taking care of the safety and economy of the extracting operation, adopt 40～45℃would be well; when the extracting time reach 60min, the extraction quantity keep stability, taking care of the economy, adopt 60min would be well; the results of uniform experiment, the more the pressure of extracting, the extracting temperature, the extracting time reach to the max, the more the extracting quantity, while in the practice operation, the pressure of extracting and the extractingtemperature can not reach the max, the pressure of extracting ought to be 25～30MPa, the extracting temperature ought to be 40～45℃, therefore, the best technology parameter would be as such: the pressure of extracting adopt 25～30MPa, the extracting temperature adopt 40～45℃, the CO₂ flow rate adopt 10～12L/h, and the extracting time 60min would be well. The results of verification test showed the technology is reasonable, reproducibility is well.There was no significant difference of extracting quantity of steam distillation adopting extracting separately or together of A (P＞0.05), taking care of the economy, adopt extracting together would be better. The best technology parameter of orthogonal experiment optimizing steam distillation technology of A: adding 5 times aquifer, no soaking, distilling 5 hours. The steam distillation technology was hard to extract the major polarity components as Ferulic acid, while the SFE-CO₂ technology can be well; and the two technologies can extract the minor polarity components as a-cyperone, while there was significant difference (P＜0.01), the SFE-CO₂ technology was more than the steam distillation technology.The extracting quantity of Ferulic acid of angelica increased with the higher of ethanol density of entrainer, the extracting quantity was the maximum when the entrainer adopting methanol, while the entrainer adopting 95% ethanol, the extracting quantity approached to methanol, taking care of the safety and economy, thus the entrainer adopt 95% ethanol would be better. The results of different technology: the extracting quantity of Ferulic acid of angelica, the SFE-CO₂ technology was the most, next was the ethanol percolating technology, and the ethanol recirculating extracting technology, and the aquifer extracting technology, the steam distillation technology can not extract out Ferulic acid.(3) The results of the single factor test of extracting clathrate of A: about the clathrate availability of clathrate variety,β-CD was the maximum, next was ethylcellulose, and silica, and carbamide, the availability ofβ-CD clathrate was obviously super to other clathrate materials; about the clathrate availability of clathrate method, the saturate solution method was the maximum, next was the grinding method, and the supersonic method; about the clathrate availability of clathrate dosage, the more the dosage ofβ-CD, the more the clathrate availability, when the clathrate dosage reach 8 times of extraction quantity, the clathrate availability keep stable; when the temperature was higher than 25℃, the clathrate availability can decrease with the temperature stepping up, thus the room temperature would be well; about the clathrate availability of clathrate time, the more the clathrate time, the more the clathrate availability, when the clathrate time reach 3 hours, the clathrate availability keep stable; in the same time, found the dosage ofβ-CD, the temperature of clathrate, the stirring time were the most important factors. The results of orthogonal experiment: the best technology parameter ofβ-CD clathrating the extraction of A, adding 8 timesβ-CD, stirring 3 hours under room temperature.the qualitative test results of extractingβ-CD. clathrate of A: about the lamellar experiment, the lamellar speckle of the extracting ligarine solution was clear, the pureβ-CD had no speckle, applying ligarine elutingβ-CD clathrate can only elute the rudimentary extracting on the surface ofβ-CD clathrate, the first time ligarine elution solution only had vague speckle, and the next time ligarine elution solution had no speckle, and only the recirculating extracting solution had the conspicuous speckle, therefore, illustrated the clathrate had formed. The scanning results of UV: the extracting solution had the maximum absorption at about 260nm. the pureβ-CD had end piece absorption, while had no absorption at about 260nm. The first time ligarine elution solution only had little peak at about 260nm, and the next timeligarine elution solution had no absorption, and only the recirculating extracting solution had the conspicuous absorption at about 260nm, therefore, further illustrated the clathrate had formed.The results of stability experiment of the extractionβ-CD clathrate of A: the CRH ofβ-CD clathrate was about 68%, thusβ-CD clathrate would keep under the situation of RH 65%, this approach to the normal Chinese drugs preparation; the expiration date ofβ-CD clathrate was about 1.26 year, this approach to the normal expiration of Chinese drug pharmaceutics.(4) Formulated the fingerprint of glycosides of Gengnian granules, marked 9 common finger peaks of glycosides of Gengnian granules. The results of reviewing methodology: the precision test results, the relative retention time and relative peak area of each common peak can keep conformity, RSD＜2%; The results of precision test, the relative retention time and relative peak area of can keep conformity in 0,2,4,6,8h, RSD＜2%, the solution can keep stable in 8 hours; the reproducibility test results, the relative retention time and relative peak area of each common peak can keep conformity, RSD＜2%. The results of resemblance calculating (complete spectra) of 10 batches sample products showed the resemblance was all more than 0.80.The results of contrasting analysis with glycosides of Gengnian granules: probably decided the origin of each finger peak.Conclusion: Applying the SFE-CO₂ technology can manifestly increase the extracting quantity and can increase variety of extracting components, can manifestly improve the extracting of volatility components, surpass the steaming distillation technology, and also surpass other extracting technology; there is intimate relationship between components extracting of medical materials and the SFE-CO₂ technology technology parameter. It is stable and feasible for the volatility components adopting modem molecule clathrate technology, its stability increases obviously, its expiration date extends obviously, achieves to medicamentous requirement. Through formulating the fingerprint of glycosides of Gengnian granules, initially probed to formulate the "complete fingerprint atlas system" of Chinese material medica, and carried out significant exploring to improve quality controlling technology of Chinese material medica. Accordingly provided foundation for the developing of modem Chinese material medica adopting advanced modern technology and diplomacy, and provided method for the modernization of Chinese drugs pharmaceutics.