| ObjectSome patients need to take no-sugar or low-sugar medicines, such as diabetes, obesity and other groups. While Ganmaoling granules (no sucrose) are developed exactly for this particular patient population. It is developed based on the Ganmaoling granules with sucrose (already listed on the market) which take the sucrose as supplementary material.According to the requirement of "Drug Registration Regulations" (current edition), the pharmaceutical and pharmacodynamics researches of Ganmaoling granules (no sucrose) had been studied. Hereinto, the pharmaceutical researches are including preparation process research as well as quality standard research.According to the determined technical parameters, preparation process and quality standards, three batches test samples were produced continuously and detected. Yield could reach over 90%, and moisture content was less than 1.5%. The products were even granules, nice outlook, of which solubility and microbial limit were qualified. The characteristic chromatograms for acetaminophen, caffeine, and chlorpheniramine maleate identified by TLC were distinct and highly specific. Also the content of acetaminophen, caffeine, chlorpheniramine maleate, and buddleoside was detected by HPLC and meets the quality standard. Also, these test samples should be stable in 6 months.Preparation Process ResearchDuring the preparation process of Ganmaoling granules (no sucrose), the formulation of Chinese medicine and chemical medicine, prescription dose, extraction, alcohol precipitation, concentration and other process of liquid extract were the same as the Ganmaoling granules with sucrose. However, the molding process of granules was changed from Wet Granulation to One Step Granulation. In the other hand, we took several indexes to evaluating different materials, including formability, moisture, solubility, bulk density, hygroscopicity, critical relative humidity and single dose. Finally, lactose was chosen as the supplementary material instead of the sucrose, and the adding dosage of lactose was determined as well. Moreover, in order to increase the patient compliance, the taste and flavour of Ganmaoling granules (no sucrose) were adjusted. Appropriate amount of steviosin was added to cover the bitter of drugs. To prevent the peppermint oil volatilizing from heating material, the peppermint oil was dissolved into ethanol and sprayed into the dry granules evenly after the granules were prepared. Hence, patients could enjoy the comfort of peppermint fragrance.The Forming Process of One Step Granulation:Chemical medicine in the prescription and lactose were well mixed in one step granulator. Then, a liquid extract made from 4 kinds of traditional Chinese herbs was sprayed on the mixture. After that, Ganmaoling granules (no sucrose) were accomplished by One Step Granulation. Also, relative density and material temperature of the liquid extract had been investigated by single factor experiment. And technological parameters in One Step Granulation were optimized by single factor and L9 (34) orthogonal experiments. Therefore, the optimal spray rate, atomizing pressure, and inlet air temperature were determined. The relative density of liquld extract was 1.22 (60℃), and spray rate was 5g/min, atomization pressure was 3bar, inlet air temperature was 75℃materiel temperature was between 50℃to 60℃. Based on those conditions, the particle forming rate could reach 96%, and the critical relative humidity was 72%. One single dose of Ganmaoling granules (no sucrose) was 2.5g, in which lactose was 1.2g (48%).In these circumstances, the preparation process of Ganmaoling granules (no sucrose) was determined. Take Caulis seu Cacumen Melicope Ptelefoliae 491g, Herba Bidentis 327g, Flos Chrysanthemi Indici 246g and Radix et Caulis Asprellae 736g. Decoct the four ingredients above with water twice,2 hours each time, filter, combine filtrates, concentrate to a thick extract with relative density of 1.10 (60℃). Cool to room temperature, add ethanol, make ethanol content reach 60-65%, stand, filter, recycle ethanol from the supernatant, then concentrate the liquid extract to relative density 1.22 (60℃). At the meantime, take paracetamol 20g, caffeine 0.4g, chlorpheniramine maleate 0.4g, lactose about 120g and steviosin about 2.5g, put into One Step Granulation machine, mix, use as the substrate material. Then the liquid extract of the four Chinese medicines was sprayed on the substrate material. Get granules about 250g from One Step Granulation, spray peppermint oil 0.2ml which was dissolved in ethanol. At last, the powder and coarse particles were sieved out, and the fine granules were packed.Quality StandardAccording to the requirement of "Drug Registration Regulations", the quality standard of Ganmaoling granules (no sucrose) had been established after optimizing the preparation process.Qualitative Examination:Acetaminophen, caffeine, and chlorpheniramine maleate were determined by TLC scanning using a mixture of chloroform, methanol, acetone, and concentrated ammonium liquid (18:3:2:0.024) as developing solvent. Locate the spots on the plate by examination under UV light at 254nm. The location and color of each principal spot in the chromatogram of acetaminophen, caffeine, and chlorpheniramine maleate corresponded to that of the principal spot in the chromatogram obtained from each relevant Standard. Quantitative Analysis: Acetaminophen, caffeine, chlorpheniramine maleate and buddleoside were quantitavely analyzed by HPLC. (1) The assay was conducted on the hypersil gold C18 column (150mm×4.6mm) of thermo electron corporation with the mobile phase of acetonitrile-0.5% phosphoric acid (6:94) and pH was adjusted to 2.10 with triethylamine, flow rate was 1.0ml·min-1, detected chlorphenamine maleate and caffeine at 264nm and acetaminophen at 244 nm. The result of methodology research indicated that the linear calibration curves of acetaminophen, caffeine, and chlorphenamine maleate were in the ranges of 0.324～1.136μg (R=0.9999),0.192～0.673μg (0.9999),0.175～0.614μg (0.9998) respectively. Their recoveries (n=9) for the assay were at 104.21%(RSD=0.92%),98.56%(RSD=1.90%),98.58% (RSD=1.30%) respectively. The results also showed that the content of acetaminophen, caffeine, and chlorpheniramine maleate in each pack was respectively set at 90.0%～110.0%,85.0%～115.0%, and 85.0%～115.0% of the labelled amount. (2) Kromasil 100-5 C18 column (150mm×4.6mm) was used with the mobile phase of methanol -0.1% phosphoric acid solution (48:52), flow rate 1.0ml·min-1 and the detection wavelengths of buddleoside was at 334nm. The result of methodology research indicated that the linear calibration curves of buddleoside showed good linear relationship with the peak area in the ranges of 0.041～0.367μg (r=1), its recoveries (n=9) for the assay were at 98.06%(RSD=1.16%). According to the results, each pack should contain buddleoside no less than 4.5mg.Stability Test:The indicators had no significant differences between the test samples in six month and zero month. Therefore, it was showed that the Ganmaoling granules (no sucrose) were stable in quality for 6 months. Pharmacodynamics ResearchIn this study, mice, rats and rabbits were test subjects. Five approaches were used in the study, which includes writhing response induced by acetic acid; swelling of hind paw in rats induced by carrageenin; delayed type hypersensitivity (DTH) in rats; capacity of mouse peritoneal macrophage phagocytosis of red blood cells; fever in rabbits induced by bacterial endotoxin. Otherwise, Lianhua Qingwen Capsule was used as reference substance.The pharmacodynamics experiment results are listed as followed:1. Ganmaoling granules (no sucrose) could obviously inhibit the pain induced by intraperitoneal injection of acetic acid to mice. The incubation periods of twisting response were prolonged to 698.82±298.16 second (P<0.001) in 10 times as human dosage group, also the times of twisting in 15 minutes were decreased (P<0.001).2. Ganmaoling granules (no sucrose) had restrictive effect on the carrageenan-induced inflammation on the rat in the post 0.5hr in 5 times as human dosage group (P< 0.05), had remarkable restrictive effect on the carrageenan-induced inflammation on the rat in the post 2hr,4 hr,6hr in 10 times as human dosage group (P< 0.05).3. Using 5 and 10 times as human dosage of Ganmaoling granules (no sucrose) for mice had certain inhibitory effect on its ear swelling and spleen index, which was induced by DNFB-induced delayed hypersensitivity, but it was not significant. While, the thymus index of mice was significantly suppressed by Ganmaoling granules (no sucrose) in 5 and 10 times as human dosage. In addition, there were no differences among inhibitory effects on ear swelling, spleen index and thymus index on mice using 5 times as human dosage of Ganmaoling granules (no sucrose).4. The capacity of mouse peritoneal macrophage phagocytosis of red blood cells was significantly improved by Ganmaoling granules (no sucrose) in 2,5,10 times as human dosage (p<0.05).5. The body temperature of rabbits in each group had increased in 1h after the 20u/k endotoxin injection. Yet, Ganmaoling granules (no sucrose) in 5 and 10 times human dosage had inhibition on the febrile effect at 1h time point (P<0.01,P<0.001). And Ganmaoling granules (no sucrose) in 5 and 10 times human dosage could inhibit the fever at 2h time point (P<0.001).6. Between Ganmaoling granules (no sucrose) and Ganmaoling granules, there was no obvious difference in the above parameters at the same dose.ConclusionOne Step Granulation using to prepare Ganmaoling granules (no sucrose) have several advantages, such as simple manufacturing process, high production speed, homogenized materials, short heating time, as well as high-yield and low power. The granules prepared by this process have good fluidity and solubility, low moisture absorption, and are easy to dissolve. Furthermore, the amount of excipient has been reduced. One single dose of Ganmaoling granules (no sucrose) is 7.5g less than that of Ganmaoling granules with sucrose. It is easy to carry, storage and consumption. And the preparations were completely free of sucrose and suitable for cold sufferers banned sucrose.The qualitative examination method for acetaminophen, caffeine, and chlorphenamine maleate in the quality standard is strong specificity, and the blank test showed no interference. In the meantime, the quantitative determination method for acetaminophen, caffeine, chlorphenamine maleate and buddleoside is accurate, sensitive and specific, also with good reproducibility. Test samples are stable in 6 months. Therefore, the established standard is applicable for the quality control of Ganmaoling granules (no sucrose).Under the conditions of this study, Ganmaoling granules with sucrose and Ganmaoling granules (no sucrose) both show antipyretic and analgesic effects. It has significant inhibition on carrageenan-induced inflammation and delayed hypersensitivity. The capacity of mouse peritoneal macrophage phagocytosis of red blood cells is significantly improved by Ganmaoling granules (no sucrose). And it also has a certain inhibition on the febrile effect of rabbits. Between Ganmaoling granules (no sucrose) and Ganmaoling granules, there is no obvious difference in the same dose.
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