| Objective: Gua Lou Mu Li Granule is a classical prescription of Zhang Zhong Jing’ s book Synopsis Golden Chamber for treating thirst,which contains Trichosanthes kirilowii Maxim and Ostrea gigas Thimberg.This study was based on Gua Lou Mu Li powder,used the modern pharmacological and chemical analysis methods,from the following aspects:prescription screening,preparation technology,quality standard and efficacy to research,which can provides experimental basis for the developing the classical into a modern new preparation for the treatment of diabetes mellitus,and research basis for application of medical institution preparations.Methods:1 The Study on Prescription Screen: 1.1 Effective dose screening:The mouse model of diabetes mellitus was established by single high dose injection of streptozotocin(stz)in mice.The model mice were randomly divided into the model control group,the positive control group,the Gua Lou Mu Li powder high,middle and low dose groups according to the blood glucose.The effective dose was screened out by continuous intragastric administration of 14 d,to detect the changes of drinking water,food intake,fasting blood glucose,fasting insulin and oral glucose tolerance test.1.2Screening of composition ratio: The mouse model of diabetes mellitus was established by single high dose injection of streptozotocin(stz)in mice.The model mice were randomly divided into the model control group,the positive control group,the radices trichosanthis group,the oyster group,the radices trichosanthis:oyster 1:2 group、the radices trichosanthis:oyster 1:1 group and the radices trichosanthis:oyster 2:1 group according to the blood glucose.The effective dose was screened out by continuous intragastric administration of 14 d,to detect the changes of drinking water,food intake,fasting blood glucose and fasting insulin.2 The Study on Preparation Process: In this experiment,Trichosanthin and Oyster were made into 200 purpose very fine powder,and the Gua Lou Mu Li Granules were prepared by wet granulation process.Investigating the type and amount of suspension based on the sedimentation volume ratio;Investigate the best drug-to-adjuvant ratio,the concentration of wetting agent and the amount of addition according to the yield,molding rate and granulation of the particles;the drying temperature is inspected according to the granules properties and molding conditions;the drying time is inspected according to the moisture of the granules after drying;granulation Process Verification;finally,the semi-finished product quality control is performed by measuring the angle of repose,bulk density and critical relative humidity of the particles.3 The Study on Quality Standard: Use the thin layer chromatography(TLC)methods to identify Trichosanthes kirilowii Maxim and Ostrea gigas Thimberg of the granules;Checking the moisture and particle sizeof the granules;determining the citrulline content in Gua Lou Mu Li granule by high performance liquid chromatography(HPLC);investigating the extraction process of trichosanthin,and use the Coomassie brilliant blue method(Bradford method)to determine the content of trichosanthin in the Gua Lou Mu Li granule;determining of calcium carbonate in Gua Lou Mu Li granule by titration.4 The Study on Efficacy: Using low doses of STZ+ high-fat diet induced T2 DM mice model,according to blood sugar,the mice were randomly divided into model control group,positive drug group,Gua Lou Mu Li granule high,medium and low group,and were given continuously for 14 d.Weight and water consumption of mice were accurately recorded daily;testing oral glucose tolerance calculate area under curve,measure fasting blood glucose,fasting insulin and calculate insulin sensitivity index,insulin resistance index,serum glycosylated protein and blood lipid levels: including total cholesterol,triglycerides,high-density lipoprotein,low-density lipoprotein,and free fatty acids;pancreas,liver,and kidney were taken for stained pathological sections to observe tissue morphology.5 The Study on the Acute Toxicity: 40 mice of KM,half male and half female,which were randomly divided into female control group,male control group,female granule group,male granule group,10 in each group.The Gua Lou Mu Li granule groups were administered by the maximum dosage method.The concentration was 0.4 g/ml,and the volume of administration was 0.4ml/10 g,twice within 24 hours.The blank group was given purified water of the same volume,the general behavior and death of mice were observed and recorded every day,the weight of mice was weighed every 2 days,and the experimental period was 14 days.Take mouse heart,liver,spleen,lung and kidney tissues to observe whether there are any abnormalities with naked eyes,and calculate the organ index to initially evaluate its acute toxicity.Results: 1 The Study on Prescription Screen: 1.1 Effective dose screening:Compared with the model group,the water intake,food intake,fasting blood glucose and oral glucose tolerance of the middle-dose(2.28g/Kg)and high-dose(4.56g/Kg)mice of Gualou Muli Powder were significantly reduced(P<0.05,P<0.01);fasting insulin increased significantly(P<0.01)1.2 Screening of composition ratio: The mice in the trichosanthes group,2:1group,1:2 group and oyster group were in poor condition and died in the late stage of gavage,while the mice in the 1:1 group were in normal condition,and compared with the model group in terms of water and food intake,fasting blood sugar was significantly reduced(P<0.05,P<0.01).2 The Study on Preparation Process: In the end,it was determined that the best drug-adjuvant ratio of Gualou Muli Powder was powder: maltodextrin:povidone k90: sodium alginate = 100: 30: 3: 3(drug powder: trichosanthin and oyster medicinal materials were made into a fine powder of 200 mesh,1:1Uniform mixing),the concentration of the wetting agent ethanol is 70%,the amount is 36%-40% of the total amount of medicinal materials and excipients,the drying temperature is 60℃,and the drying time is 1h;the granulation process verification results show that the suspension of the three batches of particles is good,indicating that the process is stable and reliable;the semi-finished product quality control experiment results show that the average angle of repose of the particles is less than 40°,the bulk density is0.382g/ml,and the critical relative humidity of the particles is 80.4.3 The Study on Quality Standard: The TLC methods can be used to identify Trichosanthes kirilowii Maxim and Ostrea gigas Thimberg,at the same time,the method of limite inspection of aconitine was built by TLC.as to glycerol trioleate,protocatechuic acid and Benzoylmesaconine,three batches of samples can show spots of the same color in the corresponding position of the chromatograms of the control medicinal materials and the control substance,and the negative samples have no interference at the same position;check the particle size and moisture content,the results meet the requirements of the Pharmacopoeia;The HPLC chromatographic was used to analyze the contents of citrulline,the determination conditions: Agilent 5 TC-C18 column(4.6mm× 250 mm,5 μm);injection volume 10μl;mobile phase:A(acetonitrile∶water=80∶20)-B(0.1 mol /L sodium acetate ∶acetonitrile=93∶7);detection wavelength: 254 nm;flow rate: 1m L/min;column temperature: 40℃.The result manifest that,the citrulline can show a good linear relationship between the range of 0.2~ 2.0 mg/m L,the average recovery rate of citrulline was 99.98%,the RSD was 1.38%,the content of citrulline in the Gua Lou Mu Li granules is about 5.169mg/g;The optimal extraction process of trichosanthin is determined as follows: the extraction solution is PBS,the material-to-liquid ratio is 1:30,the extraction time is 24 h,4000 r/min 4℃ centrifugation for 10 min,the precipitate is discarded,and the supernatant is added with 2 volumes of acetone.The precipitation time is 2 hours,centrifuged at 8000 r/min 4℃ for 20 min,discard the supernatant,and dissolve the precipitate with an appropriate amount of PBS solution to obtain the trichosanthin solution,the average recovery rate of trichosanthin was 98.08%,the RSD was 1.09%,the content of trichosanthin in the Gua Lou Mu Li granules is about 1.700mg/g;the average recovery rate of calcium carbonate was 100.34%,the RSD was 0.7%,the content of calcium carbonate in the Gua Lou Mu Li granules is about0.366g/g.4 The Study on Efficacy: The results of pharmacodynamic experiments showed that two weeks after the administration of Gua Lou Mu Li granules,the weight and water consumption of the mice in the metformin group and the high,medium and low dosage groups of Gua Lou Mu Li granules were significantly reduced(P<0.05,P<0.01);compared with the model group FBG,AUC,GHb,TC,TG,LDL-C,NEFA and IRI of diabetic mice in the metformin group and the high-dose and medium-dose groups of were significantly reduced(P<0.05,P<0.01),and FINS,HDL-C and ISI were significantly reduced increase(P<0.05,P<0.01);the pathological section results showed that the pathological damage of pancreas and kidney in the metformin group and the high and medium dose groups of Gua Lou Mu Li granules were improved to varying degrees.The pathological damage of the liver of the mice in the metformin group and the three dose groups of Gua Lou Mu Li granules were all improved.5 The Study on the Acute Toxicity: The maximum dose of Gua Lou Mu Li granules that given to the mice is 32 g/kg bw(equivalent to 78 times of the dose for human),there is no abnormal phenomenon in the mice after half an hour of administration.No poisoning or death occurred in the mice under continuous observation for 14 days.Compared with the blank control group,the body weight and organ index of the mice in the Gua Lou Mu Li granule group were not statistically significant,and there were no obvious abnormalities in the structure of the main organs.Conclusion: 1 The results of screening of effective doses and prescription ratios of Gua Lou Mu Li powder in this study showed that: the medium dose(2.28g/Kg)and high dose(4.56g/Kg)are the effective doses;Trichosanthin:Oyster = 1:1 is the best ratio.2 The preparation process of Gua Lou Mu Li granules is reasonable and the production operation is simple and feasible,which provides experimental basis and scientific basis for large-scale clinical production in the future.3 In this experiment,the quality control technology of Gua Lou Mu Li granules was systematically studied,and the quality control and evaluation method of oyster granules was preliminarily established.The method was simple,reproducible and specific,which provided the experimental basis for the establishment of its quality standard.4 This study preliminarily proves that Gua Lou Mu Li granules can effectively relieve thirst and improve the effects of hyperglycemia,obesity and lipid metabolism disorders of type 2 diabetic mice.And to a certain extent,it can improve the damage of islets,liver and pancreas caused by diabetes.5 In this study,the acute toxicity test showed that Gua Lou Mu Li granules had no toxicity to mice at 78 times dose,which indicated that oyster granules are safe under normal dose. |
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