| Hyperuricemia (HUA) is a purine abnormal metabolism, elevated blood levels of uric acid, which is genetic-related disease. The main cause is decreased excretion and (or) increased production of uric acid in the body. Uric acid is the final enzyme product of the conversion process of purine nucleotide bases and free, renal organic anion transporter of uric acid in the body maintain balance between reabsorption and secretion significantly. Reabsorption and secretion of uric acid imbalance is the main reason leading to hyperuricemia. Western medicine treatment of hyperuricemia effect is obvious, but most of them have serious side effects, making the clinical application has been limited. However, because traditional chinese medicine have multi-target effect in the body, playing a role in balancing between reabsorption and secretion of uric acid in the body, and have efficacy, side effects.Compound Tufuling granule is a hospital preparation of General Hospital of Guangzhou Military Command, the prescription of which was a proved effective recipe that has been used for many years. It contains Smilacis glabrea rhizoma, Achyranthis bidentatae radix, Cremstrae pseudoblulbus, Vaccriae semen, etal. Smilacis glabrea rhizoma as monarch drug, mainly contain flavonone, such as astilbin, isoastilbin, the main effective components astilbin have the effect of anti-inflammatory and diuretic. Achyranthis bidentatae radix as ministerial drug, mainly contain ketosteroid, such as β-ecdysterone, inokosterone and the main effective components β-ecdysterone promote protein synthesis, immune adjustment.Compound Tufuling granule has effects of Xiezhuo Chubi and dissipating dampness and clearing heat,which is mainly treatment for hyperuricemia, gout. Research on own experimental study has been done, makes a preliminary extraction process, quality standard research, through effective separation of flavonoids and total saponins, study the effective parts of uric acid transporters1and xanthine oxidase activity inhibition, proved that the drug in vitro had pharmacological mechanism. This work is to study processing technology, quality standard and pharmacodynamic of compound Tufuling granule.Through the animal experiments, had proved the preliminary pharmacodynamics of compound Tufuling granule, it provides experimental basis for new drug research. The work will research in pharmaceutics, quality standards and other aspects in accordance with national reporting requirements of the new Chinese medicine formulations.OBJECTIVEOptimization of the processing parameters was applied for the extraction, clarification, forming process of compound Tufuling granule, to determine the optimum precipitation process of the extract of compound Tufuling granule, achieving cost saving, feasible operation stability. To establish the quality standard of compound Tufuling granule for the quality control on astilbin, β-ecdysterone and total flavonoids. Through studying pharmacodynamics, proving new technology is feasible, which all lay the foundation for applying for new drug.METHODS1. Optimization of processing parameters for extraction of compound Tufuling granuleBox-Behnken experimental design combining with response surface methodology (RSM) was employed by the method of HPLC, UV, overall desirability of the extraction rate of astilbin and the extraction rate of total flavonoids and dry extract yield as dependent variables, with the extraction times (X1), the extraction time(X2), the amount of water (X3) as the independent variables, each level of independent variables was fitted by multiple linear regression and binomial formula fitting.2. Optimum clarification process of compound Tufuling granule with ZTC1+1-ⅡBy the method of HPLC and UV, the remaining rate of total flavonoid and astilbin were used as indexes.Based on the effect of some factors such as the concentration and dosage of the extracting solution, the temperature of the extracting solution when adding ZTC1+1-Ⅱ clarifiers A or B, whisking speed, holding time and standing time, orthogonal design was selected to decide the best clarification process with ZTC1+1-Ⅱ natural clarifying agents for compound Tufuling granule. A comparative study with alcohol sinking process of hospital preparations, through compared with the original alcohol precipitation processes by calculating Astilbin retention rate and the total flavonoids retention rate.3. Optimization of processing parameters for preparation of compound Tufuling granuleFirst, to examine the amount of material, through the particle quality inspection, hygroscopicity, liquidity, determine the dosage of auxiliary materials. And then, Box-Behnken experimental design combining with response surface methodology (RSM) was employed, overall desirability of solubility, the yield of qualified granule,the content of total flavonoid and astilbin, with the rate of the mixed excipients(dextrin:soluble starch), the quantiy times of exciepients(the mixed excipients:paste), the ethanol concentration and the drying temperature, as the independent variables.4. The quality standard of compound tufuling granuleSmilacis glabrea rhizoma, Achyranthis bidentatae radix in the pilot products was qualitatively identified by thin layer chromatographic method, with the control herbs and negative controls as the control. HPLC method was developed for the determination of astilbin with the column of AgilentHC-C18(4.6mm×250mm,5μm), acetonitrile-0.1%phosphoric acid(20:80) as mobile phase, detection wavelength at290nm, the flow rate of1ml·min-1, column temperature at30℃, β-ecdysterone with the column of AgilentHC-C18(4.6mm×250mm,5μm), acetonitrile-water(17:83) as mobile phase,detection wavelength at243nm,, the flow rate of1ml·min-1, column temperature at30℃, as the main effective components in compound Tufuling granule. UV method was developed for the determination of total flavonoid as the main effective constituents in compound Tufuling granule.5. The comparative study with Compound TuFuling granule pharmacodynamic studyThe effect of drug concentration and time was tested by CCK8method through cultured renal tubular epithelial cells (RTECs), absorption function test using cell uric acid was using detection of inhibitory effect of drugs on uric acid absorption. By comparing multiple mean using one-way analysis of variance, with different granule comparing inhibition effect of renal tubular epithelial cells. Using drugs to inhibit the xanthine oxidase (XOD) activity, reducing the xanthine generated the quantity of uric acid. Using uric acid kits uric acid product the quantity of uric acid,through different drugs inhibit xanthine oxidase activity, and then draw linear regression and calculate IC50, on a comparison of different drugs.RESULTS1. Optimum extraction technology was as following:extracted3times with9.8mL·g-1the amount of water forl.4h each time. The extraction rate of astilbin, the extraction rate of total flavonoids and dry extract yield were1.9906mg·g-1,54.4355mg·g-1,8.5581%g·g-1, respectively. The relative deviation between the observed and predicted values was1.54%. Compared with the original extraction process, extraction yield and extract rate of extraction process increases, and the new extraction process is economical and feasible2. The optimal process was condensing the extract solution to1:5(herb:herb solution), and the dosages of ZTC1+1-Ⅲ were6%(1%A),3%(1%B)of the extracting solution,the whisking speed was100·min-1, and standing time was24h. The remaining rate of total flavonoid, the remaining rate of astilbin and over all desirability were88.06%and85%,86.62, respectively.3. The samples were extracted by concentrating the filtrate to relative density of a thick paste about1.15～1.17, the paste mixed with excipients (soluble starch:dextrin:=1.5:1) was1:2, ethanol concentration was70%, the drying temperature was70℃.Under the above conditions, solubility is3.99min, the qualified rate of predictive value of90.34%, astilbin content of predictive value is0.0638%g·g-1, prediction value of total flavonoid content is2.0786g·g-1, predictive value of OD is0.894.4. Determined Smilacis glabrea rhizoma, Achyranthis bidentatae radix in ploit products of thin layer identification method and HPLC determination of the content of astilbin and β-ecdysterone, formulated the content limits of astilbin and β-ecdysterone, the content limits of astilbin not less than0.7374mg·g-1, β-ecdysterone content not less than0.0943mg·g-1.The determination of content of flavone by UV method, make the content of total flavonoids in limit range, flavonoids content not less than12.356mg·g-1.5. Compound tufuling granule(1,2) high concentration group (100,50g·L-1) had different degrees of inhibition on cell growth, low concentration group (25,10,5g·L-1) on the cell growth activity had no significant effect. At low concentration gradient range of (25,20,15,10,5g·L-1), uric acid absorption inhibition degree of compound tufuling granule1is higher than the effect of compound tufuling granule2. Enzyme activity test showed that the inhibition rate of IC50compound tufuling granule1and2, xanthine oxidase values were83.59μg·m-1,94.5μg·ml-1, respectively.CONCLUSIONSStudyed the extraction process of Compound Tufuling granule, through investigating the extraction time, extraction times and the amount of water using Box-Behnken response surface methodology, while introducing "normalized value" of the multi-index for processing, to determine the optimal value of each factor, the test has a better predictive model to optimize the extraction process of Compound Tufuling granule. After the experiment quantitative and the system of composite score index,using the orthogonal experiment determined the better refining process through introducing different factors and levels, proven results of the experiment show that it is stable and feasible, the preparation of Compound Tufuling granule have better moisture resistance. ZTC1+1-Ⅱ natural clarify agents can be used as clarification process in Compound Tufuling Granule extract.Four factors and five levels of central composite design-response surface method optimizes formation technology, the preparation of the resulting particle size is moderate, uniform appearancel, results of verification test also show that a reliable mathematical model has good predictability. Establishment of a viable and stable quality standard, through a preliminary comparative study pharmacodynamics, process optimization proved that it is feasible and stable. It can be used to preclinical study.The new preparation technology of Compound Tufuling granule provides basic data for new drug application. |
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