CA2710862C

CA2710862C - Pharmaceutical composition for treating diabetes and preparation method thereof

Info

Publication number: CA2710862C
Application number: CA2710862A
Authority: CA
Inventors: Jie Pan; Jipeng Chen; Fei Hong; Zhiqiang Lin
Original assignee: Zhangzhou Pientzehuang Pharmaceutical Co Ltd
Current assignee: Zhangzhou Pientzehuang Pharmaceutical Co Ltd
Priority date: 2008-01-30
Filing date: 2008-11-14
Publication date: 2019-02-26
Anticipated expiration: 2028-11-14
Also published as: CA2710862A1; CN101496829A; GB201009713D0; GB2469220B; CN101496829B; GB2469220A; WO2009100605A1

Abstract

The present invention declares a pharmaceutical composition for treating diabetes, said pharmaceutical composition mainly comprises Feculae Bombycis and Radix Glycyrrhizae. The present invention declares also declares preparation method and quality control method of said pharmaceutical composition. Said pharmaceutical composition can reduce blood sugar as well as relieve intestinal tympanites.

Description

PHARMACEUTICAL COMPOSITION FOR TREATING DIABETES AND
PREPARATION METHOD THEREOF
FIELD OF THE INVENTION
The present invention relates to a pharmaceutical composition and its preparation method, especially relates to a pharmaceutical composition for treating diabetes, and its preparation method and quality control method.
BACKGROUD OF THE INVENTION
Incidence of Diabetes (diabetes) becomes increasingly in china and abroad, which seriously harms people's health and even their lives. At present, it is understood that there are more than 200 million diabetics in the worldwide, and 85% of them are elder diabetics. There are 30 million diabetics in china, only second to the United States, but increasing at an annual rate of 750,000 new patients. Therefore, diabetes has belonged to one of major difficult diseases in national "95" projects, but efficacy of anti-diabetic traditional Chinese medicine is presently not satisfactory, and Acarbose and other drugs commonly used for treating diabetes can cause flatulence, borborygmus and other side effects. Consequently, it is necessary to carry out research on prevention and treatment of diabetes.
SUMMARY OF INVENTION
One purpose of present invention is to provide a pharmaceutical composition for treating diabetes; another purpose of the present invention is to provide preparation method and quality control method of this composition.
The purposes of present invention can be implemented through the following technical schemes:
Raw materials of this invention pharmaceutical composition consist of:
Feculae Bombycis (silkworm excrement) 6000-8000 parts by weight Radix Glycyrrhizae (licorice) 100-200 parts by weight.
Preferably, raw materials of this invention pharmaceutical composition consist of:
Feculae Bombycis 7150 parts by weight Radix Glycyrrhizae 137.5 parts by weight.
Preferably, raw materials of this invention pharmaceutical composition consist of:
Feculae Bombycis 6150parts by weight Radix Glycyrrhizae 187.5parts by weight.
Preferably, raw materials of this invention pharmaceutical composition consist of:
Feculae Bombycis 7850 parts by weight Radix Glycyrrhizae 117.5 parts by weight.

According to usual process by adding general adjuvant, this invention pharmaceutical composition is produced to tablet, capsule, powder, soft capsule, drop pill, honey pill, pill, granule, honey refined extract, sustained-release preparation, rapid-release preparation, controlled-release preparation, oral liquid or injection.
The preparation method of pharmaceutical composition of this invention can also comprise:
Taking Feculae Bombycis, adding 50-70% ethanol at 2-6 folds amount of Feculae .. Bombycis, soaking overnight, heating up slowly to boil, refluxing for 1-3 times, each time 0.5-1.5 hours, filtering, combining the filtrates, recover ethanol, after the ethanol is removed, heating the filtrates up to concentrate or vacuum concentrated at a temperature below 80 C until the relative density of 0.95-1.10 (measured at the temperature 55 C), then obtaining concentrated solution, adding water and heating to boiling, cooling, placing overnight, then taking supernatant A, heating up to concentrate the supernatant, or vacuum concentrating the supernatant at 80 C
to obtain the thick paste A with the relative density of 1.00-1.15 (measured at the temperature 55 C), stand it by service; taking Radix Glycyrrhizae, adding 8-15 folds amount of Radix Glycyrrhizae with water, decocting 1-3 times, each time 1-3 hours, combining the decoctions, placing overnight to precipitate, concentrating the supernatant B to the thick paste B with the relative density of 1.00-1.15 (measured at a temperature 5500), stand it by service; combining the thick paste A and the thick paste B, according to usual process by adding general adjuvant, then they are produced to tablet, capsule, powder, soft capsule, drop pill, honey pill, pill, granule, honey refined extract, sustained-release preparation, rapid-release preparation, controlled-release preparation, oral liquid or injection. or Taking Feculae Bombycis, adding 50-70% ethanol at 2-6 folds amount of Feculae Bombycis, soaking overnight, heating up slowly to boil, refluxing for .. times, each time 0.5-1.5 hours, filtering, combining the filtrates, recover ethanol, after the ethanol is removed, heating the filtrates up to concentrate or vacuum concentrated at a temperature below 80 C until the relative density of 0.95-1.10 (measured at the temperature 55 C), then obtaining concentrated solution, adding water and heating to boiling, cooling, placing overnight, then taking supernatant A, heating up to concentrate the supernatant, or vacuum concentrating the supernatant at 80 C
to obtain the thick paste A with the relative density of 1.00-1.15 (measured at the temperature 55 C), stand it by service; taking Radix Glycyrrhizae, adding 8-15 folds amount of Radix Glycyrrhizae with water, decocting 1-3 times, each time 1-3 hours, combining the decoctions, placing overnight to precipitate, concentrating the supernatant B to the thick paste B with the relative density of 1.00-1.15 (measured at a temperature 55 C), stand it by service; combining the thick paste A and the thick paste B, adding an appropriate amount of (3-cyclodextrin or micro silica, spraying drying, adding an appropriate amount of hydroxypropyl cellulose (L-HPC) or other general adjuvant, blending, granulating, according to usual process by adding general

2 _ . CA 02710862 2010-06-25 ..
_ adjuvant, then they are produced to tablet, capsule, powder, soft capsule, drop pill, honey pill, pill, granule, honey refined extract, sustained-release preparation, rapid-release preparation, controlled-release preparation, oral liquid or injection.
Preferably, the preparation method of pharmaceutical composition of this invention is:
Taking Feculae Bombycis, adding 60% ethanol at 4 folds amount of Feculae Bombycis, soaking overnight, heating up slowly to boil, refluxing for 2 times, each time 1 hour, filtering, combining the filtrates, recover ethanol, after the ethanol is removed, heating the filtrates up to concentrate or vacuum concentrated at a temperature below 80 C until the relative density of 1.01-1.06 (measured at the temperature 55 C), then obtaining concentrated solution, adding same amount water and heating to boiling, cooling, placing overnight, then taking supernatant A, heating up to concentrate the supernatant, or vacuum concentrating the supernatant at 80 C to obtain the thick paste A with the relative density of 1.06-1.15 (measured at the temperature 55 C), stand it by service; taking Radix Glycyrrhizae, adding 10 folds amount of Radix Glycyrrhizae with water, decocting 2 times, each time 2 hours, combining the decoctions, placing overnight to precipitate, concentrating the supernatant B to the thick paste B with the relative density of 1.04-1.15 (measured at a temperature 55 C), stand it by service; combining the thick paste A and the thick paste B, adding 13-cyclodextrin or micro silica with amount of 20% Feculae Bombycis dry paste , spraying drying, adding hydroxypropyl cellulose (L-HPC) in amount of 1%
drug powder, blending, according to usual process by adding general adjuvant, then they are produced to tablet, capsule, powder, soft capsule, drop pill, honey pill, pill, granule, honey refmed extract, sustained-release preparation, rapid-release preparation, controlled-release preparation, oral liquid or injection.
Quality control method of this invention pharmaceutical composition includes one or more of following identification and/or content determination:
Identification:
A. Taking 0.4-0.6g pharmaceutical composition of this invention, adding 4-6 ml anhydrous ethanol, refluxing in 40-80 C water bath for 20-40 minutes, placing and taking supernatant, evaporating it until 0.5-1.5 ml above water bath, that is test sample solution; furthermore, taking 0-sitosterol as reference substance, adding anhydrous ethanol for 0.0005-0.0015g/m1 solution, that is reference substance solution;
according to thin layer chromatography test, sucking above two solutions 0.001-0.003m1 respectively, pointing them on the same TLC plate of silica gel G
respectively, developing, taking out, drying, 18-21:0.3-0.7 of chloroform:
acetone as mobile phase, and spraying with 5% -15% phosphor-molybdic acid solution, heating in 100-110 C until visualize the chromatogram; the chromatogram produced by the test sample solution shows the same color spots as that displayed by the reference substance solution in their respectively corresponding areas;
B. Identification by high-performance liquid chromatography, Using

3 . -octadecylsilanized silica gel as packing; using methanol-(0.2mo1 / L) ammonium acetate - acetic acid = 50-70:30-50:0.5-1.5 as mobile phase; detection wavelength 250nm; theoretical plate number counted according to peak ammonium glycyrrhizinate is not less than 2000; weighing 0.005-0.015g mono-ammonium glycyrrhizinate reference compound accurately, putting it into 50m1 volumetric flask, dissolving by above mobile phase to the volume, shaking up, that is, 0.0001-0.0003g mono-ammonium glycyrrhizinate per ml, which is equivalent to 0.00019-0.00020g glycyrrhizic acid, that is reference substance solution;
taking pharmaceutical compositions of this invention 1.0-3.0g, put it into 25 ml volumetric flask, adding 18-22m1 above mobile phase, deal with ultrasound for minutes with power 250W, frequency of 50KHz, taking out and cooling, adding mobile phase to the volume, shaking up, centrifuging, taking the supernatant, that is test sample solution;
accurately sucking reference substance solution and test sample solution 0.005-0.015m1 respectively, injecting into liquid chromatogram instrument, test sample should be presented the same retention time peaks with the reference substance.
Content determination:
An applicability test of chromatogram condition and system Using octadecylsilanized silica gel as packing; using a solution with acetonitrile-dimethyl formamide-sodium acetate(0.025mol/L)= 15-25: 0.1-1: 75-85 as mobile phase; detection wavelength is 390nm, theoretical plate number counted according to peak Pipecolic acid is not less than 2500;
Weighing 0.004-0.006g Pipecolic acid reference compound accurately, put it into 50m1 volumetric flask, adding water to dilute to the volume, shaking up, measuring 0.5-1.5m1 the said solution accurately, putting it into 10m1 test tube with plug, adding 0.5-1.5m1 water, adding 1-3m1 2,4-acetonitrile dinitrofluorobenzene solution(0.8%) and 1-3m1 sodium bicarbonate solution(0.5mol/L), heating up in 60 C water bath for 0.5-1.5 hours, taking out, cooling, and transferring into 10m1 volumetric flask, washing container with phosphate buffer solution (0.2moUL, pH7.0) by several times, combining the washing solution into volumetric flask, adding phosphate buffer solution to the volume, shaking up, that is reference compound solution;
weighing 0.3-0.5g the pharmaceutical composition of present invention accurately, placing it in 10m1 test tube with plug, adding water, shaking up fully, heating up in 50-70 C water bath for 20-40 minutes, taking out, cooling, and transferring into 10m1 volumetric flask, washing container with water by several times, combining the washing solution into volumetric flask and adding water to volume, and shaking up;
centrifuging 12000 mm for 5-15 minutes, measuring 1-3m1 the supernatant accurately, placing it into 10m1 test tube with plug, adding 1-3m1 2,4-acetonitrile dinitrofluorobenzene solution(0.8%) and 1-3m1 sodium bicarbonate solution(0.5moUL), heating up in 50-70 C water bath for 0.5-1.5 hours, taking out, cooling, and transferring into 10m1 volumetric flask, washing container with

4 phosphate buffer solution (0.2mol/L, pH7.0) by several times, combining the washing solution into volumetric flask, adding phosphate buffer solution to the volume, shaking up, that is test sample solution, accurately sucking 0.004-0.006m1 reference substance solution and 0.005-0.015m1 test sample solution respectively, injecting into liquid chromatogram instrument, determining, that is; Feculae Bombycis content in the pharmaceutical composition of this invention in dosage per day, counted according to Pipecolic acid (C6H1 IN02), is not less than 0.0001-0.0003g.
Preferably, Quality control method of this invention pharmaceutical composition includes one or more of following identification and/or content determination:

Identification:
A. Taking 0.5g pharmaceutical composition of this invention, adding 5 ml anhydrous ethanol, refluxing in 60 C water bath for 30 minutes, placing and taking supernatant, evaporating it until 1 ml above water bath, that is test sample solution;
furthermore, taking 13-sitosterol as reference substance, adding anhydrous ethanol for 0.001g/m1 solution, that is reference substance solution; according to thin layer chromatography test, sucking above two solutions 0.002m1 respectively, pointing them on the same TLC plate of silica gel G respectively, developing, taking out, drying, 19.5:0.5 of chloroform: acetone as mobile phase, and spraying with 10%
phosphor-molybdic acid solution, heating in 105 C until visualize the chromatogram;
the chromatogram produced by the test sample solution shows the same color spots as that displayed by the reference substance solution in their respectively corresponding areas;
B. Identification by high-performance liquid chromatography, Using octadecylsilanized silica gel as packing; using methanol-(0.2mo1 / L) ammonium acetate - acetic acid = 60:40:1 as mobile phase; detection wavelength 250nm;
theoretical plate number counted according to peak ammonium glycyrrhizinate is not less than 2000; weighing 0.01g mono-ammonium glycyrrhizinate reference compound accurately, putting it into 50m1 volumetric flask, dissolving by above mobile phase to the volume, shaking up, that is, 0.0002g mono-ammonium glycyrrhizinate per ml, which is equivalent to 0.0001959g glycyrrhizic acid, that is reference substance solution;
taking pharmaceutical compositions of this invention 2g, putting it into 25 ml volumetric flask, adding 20m1 above mobile phase, deal with ultrasound for 30 minutes with power 250W, frequency of 50KHz, taking out and cooling, adding mobile phase to the volume, shaking up, centrifuging, taking the supernatant, that is test sample solution;
accurately sucking reference substance solution and test sample solution 0.01m1 respectively, injecting into liquid chromatogram instrument, test sample should be presented the same retention time peaks with the reference substance.

5 Content determination:
An applicability test of chromatogram condition and system Using octadecylsilanized silica gel as packing; using a solution with acetonitrile-dimethyl formamide-sodium acetate(0.025mol/L)= 21:0.5:79 as mobile phase; detection wavelength is 390nm, theoretical plate number counted according to peak Pipecolic acid is not less than 2500;
Weighing 0.01g Pipecolic acid reference compound accurately, putting it into 100m1 volumetric flask, adding water to dilute to the volume, shaking up, measuring 1m1 the said solution accurately, putting it into 10m1 test tube with plug, adding lml water, adding 2m1 2,4-acetonitrile dinitrofluorobenzene solution(0.8%) and 2m1 sodium bicarbonate solution(0.5mol/L), heating up in 60 C water bath for 1 hour, taking out, cooling, and transferring into 10m1 volumetric flask, washing container with phosphate buffer solution (0.2mol/L, pH7.0) by 3 times, combining the washing solution into volumetric flask, adding phosphate buffer solution to the volume, shaking up, that is reference compound solution;
weighing 0.4g the pharmaceutical composition of the present invention accurately, placing it in 10m1 test tube with plug, adding 5m1 water, shaking up fully, heating up in 60 C water bath for 30 minutes, taking out, cooling, and transferring into 10m1 volumetric flask, washing container with water by 3 times, combining the washing solution into volumetric flask and adding water to volume, and shaking up;
centrifuging 12000 rpm for 10 minutes, measuring 2m1 the supernatant accurately, placing it into 10m1 test tube with plug, adding 2m1 2,4-acetonitrile dinitrofluorobenzene solution(0.8%) and 2m1 sodium bicarbonate solution(0.5mol/L), heating up in 60 C water bath for 1 hour, taking out, cooling, and transferring into 10m1 volumetric flask, washing container with phosphate buffer solution (0.2mol/L, pH7.0) by 3 times, combining the washing solution into volumetric flask, adding phosphate buffer solution to the volume, shaking up, that is test sample solution, Accurately sucking 0.005m1 reference substance solution and 0.01m1 test sample solution respectively, injecting into liquid chromatogram instrument, determining, that is; Feculae Bombycis content in the pharmaceutical composition of this invention in dosage per day, counted according to Pipecolic acid (C61111NO2), is not less than 0.0002g.
In this invention, said relationship between weight parts and volume parts is g / ml.
Quality control methods of pharmaceutical compositions of this invention can be applied to a variety of dosage forms of compositions, such as tablets, capsules, powders, soft capsules, dipping pills, honey pills, granules, honey refining pastes, slow-release preparations, rapid-release preparations, controlled-release preparations, oral liquid preparations, or injection preparations, and other clinically acceptable dosage forms. Because different dosage forms of preparations contain same considerable amount of crude drug, in quality control of various dosage forms, selected samples can be converted into relatively uniform dose of crude drug congruously. Preparation per unit is corresponding to amount of crude drug 65g (daily dose) in the quality control method, and it can also be preparation per tablet, per piece, per injection, per pill, and so on.

6 = CA 02710862 2010-06-25 , DESCRIPTION OF THE DRAWINGS
Figure 1 is a graph depicting influence of MC for blood glucose of normal mice after sucrose feeding;
Figure 2 is a graph depicting influence of MC for blood glucose of normal mice after starch feeding;
Figure 3 is a graph depicting influence of MC for blood glucose of alloxan-induced hyperglycemic mice after sucrose feeding;
Figure 4 is a graph depicting influence of MC for blood glucose of alloxan-induced hyperglycemic mice after starch feeding;
Figure 5 is a graph depicting influence of MC for blood glucose of healthy volunteers after steamed bread feeding;
Figure 6 is a chart depicting influence of MC for urine glucose of alloxan-induced hyperglycemic rats;
Figure 7 is a chart depicting influence of MC for kidney weight/body mass index of alloxan-induced hyperglycemic rats;
Figure 8 is a chart depicting kidney, glomerulus and renal tubules of normal rats show no lesion X 50;
Figure 9 is a chart depicting that renal tubules epithelial cells of some alloxan-induced hyperglycemic rats group became swell with vacuolar appearance X 50;
Figure 10 is a chart depicting renal tubules of MC rat group rats show no significant lesion X50.
Experiments prove that pharmaceutical composition of this invention is better than raw material drugs used respectively. In addition to a hypoglycemic effect, two drugs combined have a synergistic effect, which is to improve on intestinal tympanites.
After administrating this pharmaceutical composition for 4 weeks chronically, it can significantly improve the alloxan-induced hyperglycemic rats on common symptoms of polydipsia or polyphagia; decrease blood glucose and serum fructosamine significantly; decrease serum cholesterol markedly and also triglyceride levels;
decrease serum NAG-ase activity markedly, and explain to improve on microvascular complications; decrease sorbitol content in sciatic nerve markedly, and clue to improve on chronic neuropathy of diabetes; increase GSH content in red blood cell, and enhance body's antioxidant capacity; lower kidney weight and body mass index markedly, reduce serum creatinine levels, relief glycogen deposition in renal tubular epithelial cells; in addition, inhibit a-glucosidase activity of the pharmaceutical compositions of this invention in vitro is stronger, while free for a-amylase.
It explain the pharmaceutical compositions of this invention can reduce blood sugar elevation induced by disaccharide hydrolysis absorb into blood, while the incidence of gastrointestinal side effects is lower than similar products.

7 =
The following experiments and examples are for further explanation, but not limiting the present invention.
The following experiments 1-8 use all following test medicines and experimental animals.
Test medicines:
1. Extract powder of the said pharmaceutical composition (obtain in experiment 1), abbreviated as MC, offered by Institute of Materia Medica Chinese Academy of Medical Sciences, batch number: 96102, per gram of extract powder is equivalent to 16.7g crude drug; MC extract powder dissolved with water; 2. Acarbose:
produced by Bayer pharmaceutical, Germany, batch number: 264086D.
Animals:
Kunming mice, Beijing animal quality control file number (1994), No. 029;
Wistar rats, Beijing animal quality control file number (1994), No. 030, were both purchased from breeding plants of Chinese Academy of Medical Sciences Institute, mice 22-25g, rat 180-250g, Sex: Male, animal number in each group: 10.
Alloxan-induced hyperglycemic mouse and rat models: normal animals are injected alloxan intravenously (mice 90-100 mg / kg, rats 45-50 mg / kg), predicted blood glucose (glucose oxidase method) after administering 72 hours, animals which are their blood glucose values over 11.1 mmol / L are selected for experiments.
EXPERIMENT 1: influence of MC for blood glucose elevation of normal mice after sucrose feeding Normal mice are divided into 5 groups, 10 mice each group, are fasted overnight before the experiment, a group of oral sucrose solution 4.0 g / kg as a control group, a group of oral sucrose and Acarbose 10 mg / kg as the positive control group.
The remaining three groups respectively, with oral sucrose and different doses of MC 0.45 g / kg, 0.9 g / kg, 1.8 g / kg. Determine blood glucose values at 0 min and 30 min, 60 min, 120min after the administration, results are shown in Figure 1, Table 1:
Table 1. Influence of MC for Peak blood glucose, Peak time and AUC of normal mice after sucrose feeding (mean SD, n=10) Group Peak blood glucose Peak time ( min. ) AUC ( mmol/L .hr ) ( mmol/L ) Control 9.0 1.6 30 12.9 2.8 MC 0.45 7.6 0.6 60 12.2 1.9 MC O. 9 6. 8 + 1. 9 * 60 10. 6 3. 0 MC 1. 8 5. 4+ 1. 3 ** 60 9. 2 2. 2 **
Acarbose 5.0 1.6 *** 120 9.2 + 2.7 **

8 AUC is area under blood glucose curve; compared with control group *
P<0.05,** P<0.01, *** p<0.001; MC dose is g/kg.
Experimental results show that, MC can reduce blood glucose AUC (area under curve) of normal mice after sucrose feeding, furthermore the peak blood glucose decreased significantly and shift back, which basically identical for the role of Acarbose, and show a certain dose-effect relationship.
EXPERIMENT 2: influence of MC for blood glucose elevation of normal mice after starch feeding 50 normal mice are divided into five groups, 10 mice each group, are fasted overnight, a group of soluble starch 3.0 g / kg by gavage as a control group, a group of oral sucrose and Acarbose 10 mg / kg as the positive control group. The remaining three groups respectively, with oral sucrose and different doses of MC (0.45 g / kg, 0.9 g / kg, 1.8 g / kg). Determine blood glucose values at 0 min and 30 min, 60 mm, 120min after the administration, results are shown in Figure 2, Table 2:
Table 2. Influence of MC for Peak blood glucose, Peak time and AUC of normal mice after starch feeding (mean SD, n=10) Group Peak blood glucose Peak time( mm.) AUC( mmol/L .hr ) ( mmol/L ) Control 9. 4 2. 1 30 12. 0 2. 6 MC 0.45 6.8 1.4 ** 60 10.6 1.6 MC O. 9 6. 2 + O. 6 ** 60 10. 3 1. 6 MC 1.8 4.4 0.4 *** 120 8.3 + 1.4 ***
Acarbose 4. 2 0. 5 *** 30 8. 1 + 1. 1 ***
AUC is area under blood glucose curve; compared with control group *
13<0.05,** P<0.01, *** p<0.001; MC dose is g/kg.
Results show that, MC can make the peak blood glucose decreased significantly and shift back after of normal mice after starch feeding, and reduce blood glucose AUC markedly, and show a certain dose-effect relationship.
EXPERIMENT 3: influence of MC for blood glucose elevation of normal mice after glucose feeding

9 . .
Normal mice are divided into 4 groups, 10 mice each group, are fasted overnight, a group of glucose 4.0 g / kg by gavage as a control group, a group of glucose and Acarbose 10 mg / kg by gavage as the positive control group. The remaining two groups respectively, with glucose and different doses of MC (0.45 g / kg, 1.8 g / kg) by gavage. Determine blood glucose values at 0 min and 30 min, 60 min, 120min after the administration, results are shown in Table 3:
Table 3. Influence of MC for Peak blood glucose, Peak time and AUC of normal mice after glucose feeding (mean SD, n=10) Group Peak blood glucose Peak time ( min. ) AUC( mmol/L . hr) ( mmol/L ) Control 10. 2 1. 9 30 14. 6 2. 1 MC 0.45 9.7 1.3 30 14.2 1.8 MC 1. 8 9. 4 1. 9 30 14. 8 1. 7 Acarbose 10.7 2.4 30 15.1 2.6 AUC is area under blood glucose curve; MC dose is g/kg.
Results show that, MC is identical for Acarboset, has no significant influence for blood glucose elevation of normal mice after glucose feeding. It explains that this composition does not directly affect intestinal absorption for glucose.
EXPERIMENT 4: Influence of MC for blood glucose elevation of alloxan-induced hyperglycemic mice after sucrose feeding Alloxan-induced hyperglycemic mice are divided into 5 groups, 10 mice each group, are fasted overnight, a group of sucrose 4.0 g / kg by gavage as a control group, a group of sucrose and Acarbose 10 mg / kg by gavage as the positive control group.
The remaining three groups respectively, with sucrose and different doses of MC (0.6 g / kg, 1.2 g / kg, 1.8 g / kg) by gavage. Determine blood glucose values at 0 min and 30 min, 60 min, 120min after the administration, results are shown in Figure 3, Table 4:
Table 4. Influence of MC for Peak blood glucose, Peak time and AUC of alloxan-induced hyperglycemic mice after sucrose feeding (mean SD, n=10) . CA 02710862 2010-06-25 . .
Group Peak blood glucose Peak time(min.) AUC(mmol/L.hr ) ( mmol/L ) Control 22.6 1.7 30 39.0 4.2 MC 0.6 19.3 2.7 ** 60 34.2 + 4.8 *
MC 1.2 17.1+1.3 *** 60 30. 8 2. 8 ***
MC 1.8 14.8 1.8 *** 60 28.2+ 4.8 ***
Acarbose 17.3 2.6 *** 60 30.7+ 4.3 ***
AUC is area under blood glucose curve; compared with control group *
P<0.05,** P<0.01, *** p<0.001; MC dose is g/kg.
Experimental results show that, different doses of MC can significantly reduce blood glucose elevation of alloxan-induced hyperglycemic mice after sucrose feeding, reduce blood glucose AUC significantly, make peak blood glucose value shift back, MC is identical for Acarbose at effect.
EXPERIMENT 5: Influence of MC for blood glucose elevation of alloxan-induced hyperglycemic mice after starch feeding Alloxan-induced hyperglycemic mice are divided into 5 groups, 10 mice each group, are fasted overnight, a group of starch 3.0 g / kg by gavage as a control group, a group of starch and Acarbose 10 mg / kg as the positive control group. The remaining three groups respectively, with starch and different doses of MC
(0.6 g / kg, 1.2 g / kg, 1.8 g / kg). Determine blood glucose values at 0 min and 30 min, 60 min, 120min after the administration, results are shown in Figure 4, Table 5:
Table 5. Influence of MC for Peak blood glucose, Peak time and AUC of alloxan-induced hyperglycemic mice after starch feeding (mean SD, n=10) Group Peak blood glucose Peak time(min.) ( mmol/L ) AUC( mmol/L
.hr ) -Group 24.4 1.3 30 40.6 + 4.4 Control 23.0 2.9 60 36.6 5.9 MC 0.6 21.3 + 3.1 ** 60 35.9 4.6 *
MC 1.2 19.3+2.2 *** 60 32.9 3.9 ***
MC 1.8 17.0+2.9 *** 60 31.3+5.9 ***

AUC is area under blood glucose curve; compared with control group * P<0.05,**

P<0.01, *** p<0.001; MC dose is g/kg.
Experimental results show that, different doses of MC can inhibit blood glucose elevation of alloxan-induced hyperglycemic mice after starch feeding, reduce blood glucose AUC, make peak blood glucose value shift back.

EXPERIMENT 6: Influence of MC for blood glucose of healthy volunteers after steamed bread feeding 7 volunteers (the Institute staff), 2 males and 5 females, aged 25-62 years.
In first test, after fasting, each person ate 100 g steamed bread (offered by canteen of the Institute) as self-control; in second test, randomly, 4 volunteers take MC 1.5 g / person, 3 persons take MC 3.0 g / person, and ate 100 g steamed bread at one time, Determine blood glucose values at fasting (0 min) and 30 min, 60 min, 120 min after ate steamed .. bread. Interval of two experiments is 1 week, and the results shown in Figure 5 and Table 6.
Table 6. Influence of MC for Peak blood glucose, Peak time and AUC of healthy volunteers after steamed bread feeding (mean SD ) High dose (3. 0 g/person) Low dose( 1.5 g/ person) Before After Before After administration administration administration administration Peak blood glucose 6.8 1.4 4. 6 0. 5 * 6.5 1.5 5.4 0.9 *
( mmol/L ) Peak time (mm.) 30 60 30 60 AUC(mmol/L. hr) 12.2 1.9 8.8 0.54 11.5 3.1 10.0 1.2 AUC is area under blood glucose curve; Compared after and before with themself *
P<0.05, iT. P ===-, 0.05 The preliminary results show, MC can reduce peak blood glucose value OF
healthy volunteers after steamed bread feeding, reduce blood glucose AUC, make blood glucose peak value shift back, and show dose-effect relationship.
EXPERIMENT 7: influence of MC for urine glucose of alloxan-induced hyperglycemic rats Three groups of alloxan-induced hyperglycemic rats, 10 rats each group. A
group ate high-sucrose diet as hyperglycemic control, the second and third groups ate high-sucrose diet with different doses of MC (equivalent to a dose of 0.6 g /
kg, 1.2 g /
kg) respectively. The fourth group is 10 normal rats as control group, ate normal diet.
After two weeks, collect urine by metabolic cages, measure urine sugar levels of 6 hours, simultaneously, collect urine of the fourth group, normal rat, measure it as a comparison, the results are shown in Figure 6.
The results show that: normal rats were not detected glucose in urine, MC can significantly reduce urine sugar levels in alloxan-induced hyperglycemic rats, and it shows a certain dose-effect relationship.
EXPERIMENT 8: Influence of MC for general conditions, blood glucose, blood lipids, fructosamine, serum N-acetyl-13-D glucosaminidase (short for NAG-ase) activity, sorbitol content in organization, red GSH content in blood cell, urea nitrogen and creatinine levels in serum, as well as influences of renal pathological changes, and so on of alloxan-induced hyperglycemic rats Three groups of alloxan-induced hyperglycemic rats, 10 rats each group. A
group ate high-sucrose diet as hyperglycemic control, the second and third group ate high-sucrose diet with different doses of MC (equivalent to a dose of 0.6 g /
kg, 1.2 g /
kg) respectively. The fourth group is 10 normal rats as control group, eat normal diet.
Observe the general condition of animals every day, and record food and water intake.
After 4 weeks, the animals were killed to take blood, crystalline lens, sciatic nerve and other organizations, measure blood glucose, blood lipids, fructosamine, serum NAG
activity, sorbitol content in organization, GSH content in red blood cell, urea nitrogen and creatinine levels in serum, and the other biochemical indicators, and observe renal pathological changes, the results are as follow:
1. Changes of food and water intake, Table 7:
Table 7. Influence of MC for food and water intake of alloxan-induced hyperglycemic rats;
(mean SD ) Group Food intake( g/rat/day ) Water intake( ml/ rat/day ) Normal rats 22.6 3.3 19.5 3.4 Hyperglycemic rats 125.3 15.1 118.6 19.6 MC 0.6 99.0 + 6.0 ** 61.2 + 11.2 ***
MC 1.2 179.8+ 12.1 *** 39.4 + 4.9 ***
Compared with hyperglycemic rats, ** P<0.01, *** P<0.001; MC dose is g/kg.
2. Influence of MC for blood glucose, fructosamine of alloxan-induced hyperglycemic rats, Table 8;
Fructosamine is an aldehyde-ketone condensation compound format through molecular rearrangements in the process of nonenzymatic glycosylation between plasma protein (albumin mainly) and glucose molecules. Since the stability of albumin concentration in the body, so serum fructosamine level is also stable relatively. It can reflect the blood glucose levels within 1-2 weeks.

Table 8. Influence of MC for blood glucose, fructosamine of alloxan-induced hyperglycemic rats (mean SD , n=10) Group blood glucose ( mmol/L ) Fructosamine no fasting fasting ( mmol/L ) Normal 5. 4 0. 2 3. 4 O. 5 1. 60 0. 12 25. 9 3. 6 16. 5 2. 1 2. 64 0. 16 Hyperglycemic rats MC 0. 6 19. 1+3. 4 *** 10. 4+3. 5 *** 2. 20 + O. 24 ***
MC 1. 2 10. 9 4. 4 *** 8. 3 + 3. 7 *** 2. 00 + O. 16 ***
Compared with hyperglycemic rats, *** P<0.001; MC dose is g/kg.
3. Influence of MC for blood lipids, Table 9:
Animals, which have high blood sugar, often have high blood lipids. Reducing blood glucose can improve high blood lipids. Blood lipids are determined by Enzyme method, kit purchased from Beijing Biosino Bio-technology & science INC.
Table 9. Influence of MC for blood lipids of alloxan-induced hyperglycemic rats;
(mean SD, n=10 ) Group Blood lipids( mmol/L ) Triglycerides Total cholesterol Normal rats 0.98 0.21 2.54 0.21 Hyperglycemic rats 1.83 1.00 2.78 0.24 MC 0.6 1.70 0.58 2.31 0.17 *
MC 1. 2 1. 16 0. 30 4 2. 44 O. 28 *
Compared with hyperglycemic rats, * P<0.05, 4 P==0.05 MC dose is g/kg 4. Influence of MC for serum NAG-ase activity, Table 10:
NAG enzyme is a lysosomal enzyme which present in renal parenchyma widely, and has closely relation with urinary albumin excretion and retinal microangiopathy degree, and it is a sensitive indicator of microangiopathy. With the microvascular disease increases, NAG activity increases.
Table 10. Influence of MC for serum NAG-ase activity of alloxan-induced hyperglycemic rats (mean SD, n=10) Group Serum NAG-ase ( iu ) = CA 02710862 2010-06-25 . .
Normal rats 48.3 5.9 Hyperglycemic rats 88. 0 28. 1 MC 0.6 67.5 17.0 MC 1.2 52.9 + 13.7 **
Compared with hyperglycemic rats, ** P<0.01; MC dose is g/kg 5. Influence of MC for sorbitol content in crystalline lens and sciatic nerve of alloxan-induced hyperglycemic rats, Table 11:
Sorbitol pathway is closely related with occurrence and development of chronic complications of diabetes. Increased level of tissue sorbitol content can directly reflect the activity metabolism level of sorbitol channel. Determine it with sodium arsenite-chromotropic acid method.
Table 11. Influence of MC for sorbitol content in crystalline lens and sciatic nerve of alloxan-induced hyperglycemic rats (mean SD, n=10 ) Group Sorbitol( mol/g tissue) Crystalline lens sciatic nerve Normal rats 1. 35 0. 12 2. 44 +
0. 23 Hyperglycemic rats 3.56 0.78 9.54 1.93 MC 0.6 3.47 0.55 5.71 + 1.02 ***
MC 1.2 3.39 0.82 4.15 + 1.11 ***
Compared with hyperglycemic rats, *** P<0.001; MC dose is g/kg 6. Influence of MC for reduced glutathione (GSH) content in red blood cell on alloxan-induced hyperglycemic rats, Table 12:
Free radical theory is one of major theories about occurrence and development of some diabetic chronic complications explained by molecular biology level. In high blood sugar state, GSH levels of red blood cell often low, it can reflect the body's ability to withstand free radical damage.
Table 12. Influence of MC for GSH in red blood cell of alloxan-induced hyperglycemic rats (mean SD, n=10) Group GSH( mmol/L ) P value Normal rats 0.95 + 0.16 Hyperglycemic 0. 61 1. 16 MC 0.6 1.02 0.19 <0.01 MC 1.2 1.322 0.32 <0.01 P value is compared with hyperglycemic rats; MC dose is g/kg . .
7. Influence of MC for kidney weight/body mass index of alloxan-induced hyperglycemic rats, Figure 7:
Diabetic kidney hypertrophy and hyperfiltration phenomenon are the earliest changes appearing in renal pathophysiology. Whether or not control diabetic kidney hypertrophy is often one of symbols that effectiveness of treatment of this disease.
8. Influence of MC for kidney serum urea nitrogen and creatinine levels of alloxan-induced hyperglycemic rats (Table 13):
Determine serum urea nitrogen and creatinine levels can speculate changes of renal function. Serum urea nitrogen and creatinine levels rise, then it reflect renal dysfunction. Method uses reagent kit of Beijing Biosino Bio-technology &
science INC.
Table 13. Influence of MC for kidney serum urea nitrogen and creatinine levels of alloxan-induced hyperglycemic rats (mean SD, n10) _____ Group Urea nitrogen(mmol/L) Creatinine ( mol/L ) Normal rats 6.64 1.36 47.74 6.19 Hyperglycemic 5. 39 0. 71 84. 86 14. 14 rats MC 0.6 5.75 0.75 76.91 23.87 MC 1.2 5.82 1.39 53. 04 13.26 *
Compared with hyperglycemic rats * P<0.01; MCdose is g/kg 9. Influence of MC for renal pathological changes of alloxan-induced hyperglycemic rats Experimental rat kidney in each group is 10% fixed with formalin, regular slices, stained with hematoxylin eosin, observed by light microscope. Kidney structure of normal rat is normal (see Figure 8), not seen in any lesions. Part of renal epithelial cells of kidney tubules of hyperglycemic rats are swollen and vacuolar, and after their intracellular glycogen has been dissolved during production process, membrane is visible clearly (see Figure 9); kidney tubules of MC rats isn't observed such changes (see Figure 10). Glycogen nephrosis causes by increasing blood glucose absorption of glomerular epithelial cells when through renal tubules. After reduction of blood glucose and urine glucose, other disorders can be reduced. Experiment shows that, MC can improve lesions of kidney tubules, while reduce blood and urine glucose.
The experimental results show that, after long-term (4 weeks) administration, MC can significantly improve on polydipsia or polyphagia and other general conditions of alloxan-induced hyperglycemic rats; decrease significantly blood glucose and serum fructosamine; decrease serum cholesterol levels significantly, reduce serum triglyceride levels; also decrease serum NAG activity significantly, which indicates that it has an improvement for microvascular complications;
reduce =
. .
sorbitol content in sciatic nerve significantly, which suggests that it can improve chronic neurological disease of diabetes; increase GSH content in red blood cell, enhance the body's antioxidant ability; also reduce kidney weight/body mass index significantly, reduce serum creatinine levels, relieve glycogen deposition of renal tubular epithelial cells.
Experimental 9: Experiment in vitro of pharmaceutical composition prepared in example 1 1. Test drugs: test sample: pharmaceutical composition prepared in example 1, short for MC; positive control drug: Acarbose, Orlistat.
2. Experimental animals: normal wistar rats, 250-300 g, used to extract intestinal a-glucosidase.
3. Methods and Results:
(1) Inhibit effect of MC for a-glucosidase With sucrose or maltose as substrate and Acarbose as a positive control, measure inhibition effect of MC for a-sucrose and maltose enzyme activity in rat intestinal, calculate IC50; with starch as substrate, Acarbose as a positive control, measure inhibition effect of MC for a-amylase activity, the results are shown in Table 14:
Table 14. inhibition in vitro IC50 ( pig/m1) of MC for a-glucosidase Sample a-sucrase a-maltase a-amylase MC 2.53 4.05 >100 Acarbose 0. 255 0. 076 0. 80 The results show that: MC has strong inhibitory activity on a-glucosidase in vitro, IC50 of a- sucrase and maltase are 2.53 lig / ml and 4.05 ttg / ml; and it has no inhibitory effect on a-amylase activity.
(2) Inhibit effect of MC for lipase activity With triolein as substrate and Orlistat as a positive control, test samples MC

response to porcine pancreatic lipase and lipoprotein lipase respectively, determine inhibitory activity of sample to lipase, by release rate of oleic acid released from triolein. The results are shown in Table 15:
Table 15. Inhibitory rate of MC for lipase activity Terminal concentration Sample ( P g/ml ) Pancreatic lipase Lipoprotein lipase Orlistat 10 100% 100%
The results show that MC almost no inhibitory effect on lipase.
4. Experimental result:

= CA 02710862 2010-06-25 Experimental results show that, samples of MC have a very strong inhibitory effect on a-sucrose and a-maltase activity, and IC50 are 2.53 itg / ml and 4.05 p.g /
ml respectively; do not inhibit a-amylase. It shows that MC only inhibit disaccharide hydrolase without inhibiting a-amylase activity, thus it greatly reduces amount of starch and oligosaccharides without fully hydrolyzed into intestinal bottom end, only part remnant disaccharide into intestinal bottom end, reduce hydrolyzed disaccharide absorbed into blood, make blood glucose elevation, their incidence of gastrointestinal side effects is lower than Acarbose. MC has no inhibitory effect on lipase, it shows that MC can not directly affect fat absorption from intestine.
The following examples can entirely achieve efficacies in above experiments.
EMBODIMENTS
EXAMPLE 1: PREPARATION OF CAPSULE
Feculae Bombycis 7.15kg Radix Glycyrrhizae 0.1375kg Taking Feculae Bombycis, adding 60% ethanol at 4 folds amount of Feculae Bombycis, soaking overnight, heating up slowly to boil, refluxing for 2 times, each time 1 hours, filtering, combining the filtrates, recovering ethanol, concentrating at a temperature 55 C, gaining concentrated solution of the relative density of 1.01-1.06, adding same amount water and heating to boiling, cooling, placing overnight, then taking supernatant A, heating the supernatant to obtain the thick paste A with the relative density of 1.06-1.15 (measured at the temperature 55 C), stand it by service;
taking Radix Glycyrrhizae, adding 10 folds amount of Radix Glycyrrhizae with water, decocting 2 times, each time 2 hours, combining the decoctions, placing overnight to precipitate, concentrating the supernatant B to the thick paste B with the relative density of 1.04-1.15 (measured at a temperature 55 C), stand it by service;
combining the thick paste A and the thick paste B, adding 0-cyclodextrin with amount of 20%
Feculae Bombycis dry paste, spraying drying, add hydroxypropyl cellulose (L-HPC) in amount of 1% drug powder, blending, drying, granulating, whole grain, putting into capsules, obtaining 1000 capsules, 2-4 tablets each time, three times daily.
EXAMPLE 2: PREPARATION OF TABLET
Feculae Bombycis 7.15kg Radix Glycyrrhizae 0.1375kg , taking above two crude drugs, according to usual process, adding general adjuvant, producing tablet.
EXAMPLE 3: PREPARATION OF GRANULE
Feculae Bombycis 6.15kg Radix Glycyrrhizae 0.1875kg , taking above two crude drugs, according to usual process, adding general adjuvant, producing granule.

=

EXAMPLE 4: PREPARATION OF PILL
Feculae Bombycis 7.85kg Radix Glycyrrhizae 0.1175kg , taking above two crude drugs, according to usual process, adding general adjuvant, producing pill.
EXAMPLE 5: PREPARATION OF HONEY REFINING PASTE
Feculae Bombycis 7.15kg Radix Glycyrrhizae 0.1375kg , taking above two crude drugs, according to usual process, adding general adjuvant, producing honey refining paste.
EXAMPLE 6: PREPARATION OF ORAL LIQUID PREPARATION
Feculae Bombycis 6.15kg Radix Glycyrrhizae 0.1875kg , taking above two crude drugs, according to usual process, adding general adjuvant, producing oral liquid preparation.
EXAMPLE 7: PREPARATION OF INJECTION
Feculae Bombycis 7.85kg Radix Glycyrrhizae 0.1175kg , taking above two crude drugs, according to usual process, adding general adjuvant, producing injection.
EXAMPLE 8: Identification and content determination methods of the pharmaceutical composition capsule Identification:
A. Taking 0.5g capsule content of this invention pharmaceutical composition prepared in Example 1, adding 5 ml anhydrous ethanol, refluxing in 60 C water bath for 30 minutes, placing and taking supernatant, evaporating it until 1 ml above water bath, that is test sample solution; furthermore, taking 0-sitosterol as reference substance, adding anhydrous ethanol for 0.001g/m1 solution, that is reference substance solution; according to thin layer chromatography test, sucking above two solutions 0.002m1 respectively, pointing them on the same TLC plate of silica gel G
respectively, developing, taking out, drying, 19.5:0.5 of chloroform: acetone as mobile phase, and spraying with 10% phosphor-molybdic acid solution, heating in 105 C until visualize the chromatogram; the chromatogram produced by the test sample solution shows the same color spots as that displayed by the reference substance solution in their respectively corresponding areas;
B. Identification by high-performance liquid chromatography, Using octadecylsilanized silica gel as packing; using methanol-(0.2m01 / L) ammonium acetate - acetic acid = 60:40:1 as mobile phase; detection wavelength 250nm;
theoretical plate number counted according to peak ammonium glycyrrhizinate is not less than 2000; weighing 0.01g mono-ammonium glycyrrhizinate reference compound . .
= CA 02710862 2010-06-25 .
accurately, putting it into 50m1 volumetric flask, dissolving by above mobile phase to the volume, shaking up, that is, 0.0002g mono-ammonium glycyrrhizinate per ml, which is equivalent to 0.0001959g glycyrrhizic acid, that is reference substance solution;
taking 2g capsule content of this invention pharmaceutical compositions prepared in Example 1, putting it into 25 ml volumetric flask, adding 20m1 above mobile phase, deal with ultrasound for 30 minutes with power 250W, frequency of 50KHz, taking out and cooling, adding mobile phase to the volume, shaking up, centrifuging, taking the supernatant, that is test sample solution;
Accurately sucking reference substance solution and test sample solution 0.005ml respectively, injecting into liquid chromatogram instrument, test sample should be presented the same retention time peaks with the reference substance.
Content determination:
An applicability test of chromatogram condition and system Using octadecylsilanized silica gel as packing; using a solution with acetonitrile-dimethyl formamide-sodium acetate(0.025mol/L)= 21:0.5:79 as mobile phase; detection wavelength is 390nm, theoretical plate number counted according to peak Pipecolic acid is not less than 2500;
Weighing 0.01g Pipecolic acid reference compound accurately, putting it into 100m1 volumetric flask, adding water to dilute to the volume, shaking up, measuring 1m1 the said solution accurately, putting it into 10m1 test tube with plug, adding 1m1 water, adding 2m1 2,4-acetonitrile dinitrofluorobenzene solution(0.8%) and 2m1 sodium bicarbonate solution(0.5mol/L), heating up in 60 C water bath for 1 hour, taking out, cooling, and transferring into 10m1 volumetric flask, washing container with phosphate buffer solution (0.2mol/L, pH7.0) by 3 times, combining the washing solution into volumetric flask, adding phosphate buffer solution to the volume, shaking up, that is reference compound solution;
accurately weighing 0.4g capsule content of this invention pharmaceutical composition prepared in Example 1, placing it in 10m1 test tube with plug, adding 5m1 water, shaking up fully, heating up in 60 C water bath for 30 minutes, taking out, cooling, and transferring into 10m1 volumetric flask, washing container with water by 3 times, combining the washing solution into volumetric flask and adding water to volume, and shaking up; centrifuging 12000 rpm for 10 minutes, measuring 2m1 the supernatant accurately, placing it into 10m1 test tube with plug, adding 2m1 2,4-acetonitrile dinitrofluorobenzene solution(0.8%) and 2m1 sodium bicarbonate solution(0.5mol/L), heating up in 60 C water bath for 1 hour, taking out, cooling, and transferring into 10m1 volumetric flask, washing container with phosphate buffer solution (0.2mol/L, pH7.0) by 3 times, combining the washing solution into volumetric flask, adding phosphate buffer solution to the volume, shaking up, that is test sample solution, Accurately sucking 0.005m1 reference substance solution and 0.01m1 test sample solution respectively, injecting into liquid chromatogram instrument, determining, that is; dosage per day for the pharmaceutical composition of this invention is 0.5g, then Feculae Bombycis content counted according to Pipecolic acid (C6H11NO2), is not less than 0.0018g.
EXAMPLE 9: Identification method of the pharmaceutical composition powder A. Taking 0.5g pharmaceutical composition powder of this invention, adding 6 ml anhydrous ethanol, refluxing in 40 C water bath for 40 minutes, placing and taking supernatant, evaporating it until 0.5 ml above water bath, that is test sample solution;
furthermore, taking 0-sitosterol as reference substance, adding anhydrous ethanol for 0.0015g/m1 solution, that is reference substance solution; according to thin layer chromatography test, sucking above two solutions 0.00 1 ml respectively, pointing them on the same TLC plate of silica gel G respectively, developing, taking out, drying, 21:0.3 of chloroform: acetone as mobile phase, and spraying with 15%
phosphor-molybdic acid solution, heating in 100 C until visualize the chromatogram;
the chromatogram produced by the test sample solution shows the same color spots as that displayed by the reference substance solution in their respectively corresponding areas;
B. Identification by high-performance liquid chromatography, Using octadecylsilanized silica gel as packing; using methanol-(0.2mo1 / L) ammonium acetate - acetic acid = 50:50:0.5 as mobile phase; detection wavelength 250nm;

theoretical plate number counted according to peak ammonium glycyrrhizinate is not less than 2000; weighing 0.005g mono-ammonium glycyrrhizinate reference compound accurately, putting it into 50m1 volumetric flask, dissolving by above mobile phase to the volume, shaking up, that is, 0.0003g mono-ammonium glycyrrhizinate per ml, which is equivalent to 0.00019g glycyrrhizic acid, that is reference substance solution;
taking 3.0g pharmaceutical composition powder of this invention, putting it into 25 ml volumetric flask, adding 18m1 above mobile phase, deal with ultrasound for minutes with power 250W, frequency of 50KHz, taking out and cooling, adding mobile phase to the volume, shaking up, centrifuging, taking the supernatant, that is test sample solution;
accurately sucking reference substance solution and test sample solution 0.015m1 respectively, injecting into liquid chromatogram instrument, test sample should be presented the same retention time peaks with the reference substance.
EXAMPLE 10: Content determination method of the pharmaceutical composition granule An applicability test of chromatogram condition and system Using octadecylsilanized silica gel as packing; using a solution with acetonitrile-dimethyl formamide-sodium acetate(0.025mo1/L)= 15:1:75 as mobile = CA 02710862 2010-06-25 phase; detection wavelength is 390nm, theoretical plate number counted according to peak Pipecolic acid is not less than 2500;
Weighing 0.006g Pipecolic acid reference compound accurately, putting it into 50m1 volumetric flask, adding water to dilute to the volume, shaking up, measuring 0.5m1 the said solution accurately, putting it into 10m1 test tube with plug, adding 1.5m1 water, adding lml 2,4-acetonitrile dinitrofluorobenzene solution(0.8%) and 3m1 sodium bicarbonate solution(0.5mol/L), heating up in 60 C water bath for 0.5 hour, taking out, cooling, and transferring into 10m1 volumetric flask, washing container with phosphate buffer solution (0.2mo1/L, pH7.0) by several times, combining the washing solution into volumetric flask, adding phosphate buffer solution to the volume, shaking up, that is reference compound solution;
accurately weighing 0.5g granule of this invention pharmaceutical composition, placing it in 10m1 test tube with plug, adding proper amount water, shaking up fully, heating up in 50 C water bath for 40 minutes, taking out, cooling, and transferring into 10m1 volumetric flask, washing container with water by several times, combining the washing solution into volumetric flask and adding water to volume, and shaking up; centrifuging 12000 rpm for 5 minutes, measuring 3m1 the supernatant accurately, placing it into 10m1 test tube with plug, adding lml 2,4-acetonitrile dinitrofluorobenzene solution(0.8%) and 3m1 sodium bicarbonate solution(0.5mol/L), heating up in 50 C water bath for 1.5 hour, taking out, cooling, and transferring into 10m1 volumetric flask, washing container with phosphate buffer solution (0.2mol/L, pH7.0) by several times, combining the washing solution into volumetric flask, adding phosphate buffer solution to the volume, shaking up, that is test sample solution, Accurately sucking 0.004m1 reference substance solution and 0.015m1 test sample solution respectively, injecting into liquid chromatogram instrument, determining, that is; dosage per day for the pharmaceutical composition of this invention is 0.5g, then Feculae Bombycis content counted according to Pipecolic acid (C6H111\102), is not less than 0.0018g.
EXAMPLE 11: Identification and content determination methods of the pharmaceutical composition tablet Identification:
A. Taking 0.5g tablet of this invention pharmaceutical composition prepared in Example 2, adding 5g anhydrous ethanol, refluxing in 60 C water bath for 30 minutes, placing and taking supernatant, evaporating it until 1 ml above water bath, that is test sample solution; furthermore, taking 0-sitosterol as reference substance, adding anhydrous ethanol for 0.001g/m1 solution, that is reference substance solution;
according to thin layer chromatography test, sucking above two solutions 0.002m1 respectively, pointing them on the same TLC plate of silica gel G
respectively, developing, taking out, drying, 19.5:0.5 of chloroform: acetone as mobile phase, and spraying with 10% phosphor-molybdic acid solution, heating in 105 C until visualize . .

. the chromatogram; the chromatogram produced by the test sample solution shows the same color spots as that displayed by the reference substance solution in their respectively corresponding areas;
Content determination:
An applicability test of chromatogram condition and system Using octadecylsilanized silica gel as packing; using a solution with acetonitrile-dimethyl formamide-sodium acetate(0.025mo1/L)= 21:0.5:79 as mobile phase; detection wavelength is 390nm, theoretical plate number counted according to peak Pipecolic acid is not less than 2500;
Weighing 0.005g Pipecolic acid reference compound accurately, putting it into 50m1 volumetric flask, adding water to dilute to the volume, shaking up, measuring 1m1 the said solution accurately, putting it into 10m1 test tube with plug, adding lml water, adding 2m1 2,4-acetonitrile dinitrofluorobenzene solution(0.8%) and 2 parts by volume of sodium bicarbonate solution(0.5mo1/L), heating up in 60 C water bath for 1 hour, taking out, cooling, and transferring into 10m1 volumetric flask, washing container with phosphate buffer solution (0.2m01/L, pH7.0) by several times, combining the washing solution into volumetric flask, adding phosphate buffer solution to the volume, shaking up, that is reference compound solution;
accurately weighing 0.4g tablet of this invention pharmaceutical composition prepared in Example 2, placing it in 10m1 test tube with plug, adding proper amount water, shaking up fully, heating up in 60 C water bath for 30 minutes, taking out, cooling, and transferring into 10m1 volumetric flask, washing container with water by several times, combining the washing solution into volumetric flask and adding water to volume, and shaking up; centrifuging 12000 rpm for 10 minutes, measuring 2m1 the supernatant accurately, placing it into 10m1 test tube with plug, adding 2m1 2,4-acetonitrile dinitrofluorobenzene solution(0.8%) and 2m1 sodium bicarbonate solution(0.5mol/L), heating up in 60 C water bath for 1 hour, taking out, cooling, and transferring into 10m1 volumetric flask, washing container with phosphate buffer solution (0.2mol/L, pH7.0) by several times, combining the washing solution into volumetric flask, adding phosphate buffer solution to the volume, shaking up, that is test sample solution, accurately sucking 0.005m1 reference substance solution and 0.01m1 test sample solution respectively, injecting into liquid chromatogram instrument, determining, that is; dosage per day for the pharmaceutical composition of this inventionis 0.5g, then Feculae Bombycis content counted according to Pipecolic acid (C6H1 INN, is not less than 0.0018g.
EXAMPLE 12: PREPARATION OF TABLET
Feculae Bombycis 7.15kg Radix Glycyrrhizae 0.1375kg Taking Feculae Bombycis, adding 60% ethanol at 4 folds amount of Feculae Bombycis, soaking overnight, heating up slowly to boil, refluxing for 2 times, each time 1 hour; filtering, combining the filtrates, recovering ethanol, concentrating at a temperature 55 C, gaining concentrated solution of the relative density of 1.01-1.06, adding same amount water and heating to boiling, cooling, placing overnight, then taking supernatant A, heating the supernatant to obtain the thick paste A with the relative density of 1.06-1.15 (measured at the temperature 55 C), stand it by service;
taking Radix Glycyrrhizae, adding 10 folds amount of Radix Glycyrrhizae with water, decocting 2 times, each time 2 hours, combining the decoctions, placing overnight to precipitate, concentrating the supernatant B to the thick paste B with the relative density of 1.04-1.15 (measured at a temperature 55 C), stand it by service;
combining the thick paste A and the thick paste B, according to usual process, adding general adjuvant, producing tablet.
EXAMPLE 13: PREPARATION OF GRANULE
Feculae Bombycis 6.15kg Radix Glycyrrhizae 0.1875kg Taking Feculae Bombycis, adding 60% ethanol at 4 folds amount of Feculae Bombycis, soaking overnight, heating up slowly to boil, refluxing for 2 times, each time 1 hour, filtering, combining the filtrates, recovering ethanol, concentrating at a temperature 55 C, gaining concentrated solution of the relative density of 1.01-1.06, adding same amount water and heating to boiling, cooling, placing overnight, then taking supernatant A, heating the supernatant to obtain the thick paste A with the relative density of 1.06-1.15 (measured at the temperature 55 C), stand it by service;
taking Radix Glycyrrhizae, adding 10 folds amount of Radix Glycyrrhizae with water, decocting 2 times, each time 2 hours, combining the decoctions, placing overnight to .. precipitate, concentrating the supernatant B to the thick paste B with the relative density of 1.04-1.15 (measured at a temperature 55 C), stand it by service;
combining the thick paste A and the thick paste B, according to usual process, adding general adjuvant, producing granule.
EXAMPLE 14: PREPARATION OF PILL
Feculae Bombycis 7.85kg Radix Glycyrrhizae 0.1175kg Taking Feculae Bombycis, adding 60% ethanol at 4 folds amount of Feculae Bombycis, soaking overnight, heating up slowly to boil, refluxing for 2 times, each time 1 hour, filtering, combining the filtrates, recovering ethanol, concentrating at a temperature 55 C, gaining concentrated solution of the relative density of 1.01-1.06, adding same amount water and heating to boiling, cooling, placing overnight, then taking supernatant A, heating the supernatant to obtain the thick paste A with the relative density of 1.06-1.15 (measured at the temperature 55 C), stand it by service;
taking Radix Glycyrrhizae, adding 10 folds amount of Radix Glycyrrhizae with water, decocting 2 times, each time 2 hours, combining the decoctions, placing overnight to precipitate, concentrating the supernatant B to the thick paste B with the relative density of 1.04-1.15 (measured at a temperature 55 C), stand it by service;
combining the thick paste A and the thick paste B, according to usual process, adding general adjuvant, producing pill.
EXAMPLE 15: PREPARATION OF HONEY REFINING PASTE
Feculae Bombycis 7.15kg Radix Glycyrrhizae 0.1375kg Taking Feculae Bombycis, adding 60% ethanol at 4 folds amount of Feculae Bombycis, soaking overnight, heating up slowly to boil, refluxing for 2 times, each . .
= CA 02710862 2010-06-25 . .
time 1 hour, filtering, combining the filtrates, recovering ethanol, concentrating at a temperature 55 C, gaining concentrated solution of the relative density of 1.01-1.06, adding same amount water and heating to boiling, cooling, placing overnight, then taking supernatant A, heating the supernatant to obtain the thick paste A with the relative density of 1.06-1.15 (measured at the temperature 55 C), stand it by service;
taking Radix Glycyrrhizae, adding 10 folds amount of Radix Glycyrrhizae with water, decocting 2 times, each time 2 hours, combining the decoctions, placing overnight to precipitate, concentrating the supernatant B to the thick paste B with the relative density of 1.04-1.15 (measured at a temperature 55 C), stand it by service;
combining the thick paste A and the thick paste B, according to usual process, adding general adjuvant, producing honey refining paste.
EXAMPLE 16: PREPARATION OF ORAL LIQUID PREPARATION
Feculae Bombycis 6.15kg Radix Glycyrrhizae 0.1875kg Taking Feculae Bombycis, adding 60% ethanol at 4 folds amount of Feculae Bombycis, soaking overnight, heating up slowly to boil, refluxing for 2 times, each time 1 hour, filtering, combining the filtrates, recovering ethanol, concentrating at a temperature 55 C, gaining concentrated solution of the relative density of 1.01-1.06, adding same amount water and heating to boiling, cooling, placing overnight, then taking supernatant A, heating the supernatant to obtain the thick paste A with the relative density of 1.06-1.15 (measured at the temperature 55 C), stand it by service;
taking Radix Glycyrrhizae, adding 10 folds amount of Radix Glycyrrhizae with water, decocting 2 times, each time 2 hours, combining the decoctions, placing overnight to precipitate, concentrating the supernatant B to the thick paste B with the relative density of 1.04-1.15 (measured at a temperature 55 C), stand it by service;
combining the thick paste A and the thick paste B, according to usual process, adding general adjuvant, producing oral liquid preparation.
EXAMPLE 17: PREPARATION OF INJECTION
Feculae Bombycis 7.85kg Radix Glycyrrhizae 0.1175kg Taking Feculae Bombycis, adding 60% ethanol at 4 folds amount of Feculae Bombycis, soaking overnight, heating up slowly to boil, refluxing for 2 times, each time 1 hour, filtering, combining the filtrates, recovering ethanol, concentrating at a temperature 55 C, gaining concentrated solution of the relative density of 1.01-1.06, adding same amount water and heating to boiling, cooling, placing overnight, then taking supernatant A, heating the supernatant to obtain the thick paste A with the relative density of 1.06-1.15 (measured at the temperature 55 C), stand it by service;
taking Radix Glycyrrhizae, adding 10 folds amount of Radix Glycyrrhizae with water, decocting 2 times, each time 2 hours, combining the decoctions, placing overnight to precipitate, concentrating the supernatant B to the thick paste B with the relative density of 1.04-1.15 (measured at a temperature 55 C), stand it by service;
combining the thick paste A and the thick paste B, according to usual process, adding general adjuvant, producing injection.

Claims

1. A pharmaceutical composition consisting of:
6000-8000 parts by weight of a material of Feculae Bombycis; and 100-200 parts by weight of a material of Radix Glycyrrhizae;
wherein said composition is prepared by the following method:
taking Feculae Bombycis, adding 50-70% ethanol at 2-6 fold the amount of Feculae Bombycis, soaking overnight, heating up slowly to boil, refluxing for 1-3 times, each time 0.5-1.5 hours, filtering, combining the filtrates, removing ethanol, after the ethanol is removed, heating the filtrates up to concentrate or vacuum concentrating the filtrates at a temperature below 80°C until the relative density of 0.95-1.10, measured at the temperature 55°C, then obtaining a concentrated solution, adding water and heating to boiling point, cooling, leave to stand overnight, then taking a supernatant A, heating up to concentrate the supernatant, or vacuum concentrating the supernatant at 80°C to obtain a thick paste A with the relative density of 1.00-1.15, measured at the temperature 55°C, let the thick paste A stand; taking Radix Glycyrrhizae, adding 8-15 fold the amount of Radix Glycyrrhizae with water, decocting 1-3 times, each time 1-3 hours, combining the decoctions, leave to stand overnight to precipitate, concentrating a supernatant B to a thick paste B
with the relative density of 1.00-1.15, measured at a temperature 55°C, let the thick paste B
stand; combining the thick paste A and the thick paste B thereby producing said composition.

2. The pharmaceutical composition of claim 1, said pharmaceutical composition consisting of:
7150 parts by weight of the material of Feculae Bombycis; and 137.5 parts by weight of the material of Radix Glycyrrhizae.

3. The pharmaceutical composition of claim 1, said pharmaceutical composition consisting of:
6150 parts by weight of the material of Feculae Bombycis; and 187.5 parts by weight of the material of Radix Glycyrrhizae.

4. The pharmaceutical composition of claim 1, said pharmaceutical composition consisting of:
7850 parts by weight of the material of Feculae Bombycis 117.5 parts by weight of the material of Radix Glycyrrhizae.

5. The pharmaceutical composition of any one of claims 1 to 4, wherein said method is:
taking Feculae Bombycis, adding 60% ethanol at 4 folds amount of Feculae Bombycis, soaking overnight, heating up slowly to boil, refluxing for 2 times, each time 1 hour, filtering, combining the filtrates, removing ethanol, after the ethanol is removed, heating the filtrates up to concentrate or vacuum concentrating the filtrates at a temperature below 80°C until the relative density of 1.01-1.06 measured at the temperature 55°C, then obtaining concentrated solution, adding same amount water and heating to boiling, cooling, leaving to stand overnight, then taking supernatant A, heating up to concentrate the supernatant, or vacuum concentrating at 80°C to obtain the thick paste A with the relative density of 1.06-1.15, measured at the temperature 55°C; taking Radix Glycyrrhizae, adding 10 folds amount of Radix Glycyrrhizae with water, decocting 2 times, each time 2 hours, combining the decoctions, leaving to stand overnight to allow precipitation, concentrating the supernatant B to the thick paste B with the relative density of 1.04-1.15, measured at a temperature 55°C;
combining the thick paste A and the thick paste B.

6. The pharmaceutical composition of any one of claims 1 to 5, further comprising a general adjuvant.

7. The pharmaceutical composition of claim 6 formulated in the form of a tablet, capsule, powder, soft capsule, drop pill, honey pill, pill, granule, honey refilled extract, sustained-release preparation, rapid-release preparation, controlled release preparation, oral liquid preparation or injection preparation.

8. A method for controlling quality of the pharmaceutical composition of any one of claims 1 to 5, wherein said method includes the following identification and content determination steps ldentification:

A Adding 0.4-0.6 g of the pharmaceutical composition in 4-6 mL anhydrous ethanol to produce a solution, refluxing the solution at 40-80°C in a water bath for 20-40 minutes and then letting the solution standing. taking a supernatant from the solution and evaporating the supernatant above a water bath until 0.5-1.5 mL is left.
to provide a test sample solution;
furthermore, taking .beta.-sitosterol as a reference substance which is added to anhydrous ethanol to provide a reference substance solution having a concentration of 0.0005-0.001 5 g/mL;
performing a thin layer chromatography test by sucking 0.001-0.003 mL of said test sample solution and 0.001-0.003 mL of said reference substance solution, spotting them on a same TLC (thin layer chromatography) silica gel plate and developing them by using a developing solvent composed of chloroform and acetone at a weight ratio of 18-21 : 0.3-0.7, then drying the silica gel plate and spraying it with 5% -15%
phosphor-molybdic acid solution. followed by heating the silica gel plate at 110°C until a chromatogram is visualized; the chromatogram produced by the test sample solution shows the same color spots as that displayed by the reference substance solution;
B identification by high-performance liquid chromatography, using octadecylsilanized silica gel as a packing; using a mixture of methanol, ammonium acetate of 0.2mol/I. and acetic acid at a weight ratio of 50-70 : 30-50 : 0.5-1.5 as a mobile phase; detection wavelength is 250nm; theoretical plate number counted according to peak ammonium glycyrrhizinate is not less than 2000;
weighing 0.005-0.015 g mono-ammonium glycyrrhizinate accurately, dissolving it with said mobile phase in a volumetric flask to 50 mL and shaking up. to provide a reference substance solution containing mono-ammonium glycyrrhizinate at 0.0001-0.0003 g/mL, which is equivalent to 0.00019-0.00020 g/mL of glycyrrhizic acid;
taking 1.0-3.0 g of said pharmaceutical composition, putting it into a volumetric flask of 25 mL, adding 18-22 mL of said mobile phase into the flask to produce a mixture, exposing the mixture to ultrasound treatment for 20-40 minutes with power 250W

and frequency of 50KHz, then cooling the mixture, adding the mobile phase to the mixture until the volume reaches 25mL, shaking up the mixture, followed by centrifuging the mixture, and then taking a supernatant from the centrifuged mixture for using as a test sample solution, accurately sucking 0.005-0.015 ml, of the reference substance solution and 0.005-0.015 mL of the test sample solution and injecting them into a liquid chromatogram instrument. ensuring that the test sample solution should show a chromatographic peak having a same retention time as that of the reference substance solution;
Content determination:
An applicability test of chromatogram condition and system using octadecylsilanized silica gel as a packing; using a mixture of acetonitrile, dimethyl formamide and sodium acetate of 0.025mol/L at a weight ratio of 15-25:
0.1-1: 75-85 as a mobile phase;
detection wavelength is 390nm, and the number of theoretical plates is not less than 2500 counted according to the peak of pipecolic acid;
accurately weighing 0.004-0.006 g of pipecolic acid as reference compound, putting it into a first volumetric flask, adding water to dilute to 50 mL and shaking up to produce a first solution, measuring 0.5-1.5 mL of the first solution accurately and putting it into a first plugged test tube with a volume of 10 mL, adding 0.5-1.5 mL
water, 1-3 mL 2,4-acetonitrile dinitrofluorobenzene solution having a concentration of 0.8% by weight and 1-3 mL sodium bicarbonate solution having a concentration of 0.5mol/L to the tube to produce a second solution, heating the second solution at 60 C in a water bath for 0.5-1.5 hours, taking out the second solution in the tube from the water bath, cooling the second solution, and transferring the second solution from the tube into a second volumetric flask of 10mL. washing the tube with a phosphate hurler solution having a concentration of 0.2mol/L and a pH value of 7.0 for several times, collecting the phosphate buffer solution after each of said washing and combining it into the second volumetric flask, adding a further amount of the phosphate buffer solution to the second volumetric flask until the volume reaches 10mL, and then shaking the second volumetric flask to provide a reference compound solution;
weighing 0.3-0.5 g of the pharmaceutical composition accurately, placing it in a second plugged test tube with a volume of 10 mL, adding 5 mL water in the tube and shaking it up fully to produce a third solution, heating up the tube containing the third solution at 50-70°C in a water bath for 20-40 minutes, taking out the tube and cooling the third solution, and transferring the third solution from the tube into a third volumetric flask of 10 mL, washing the tube with water for several times, collecting the water after each of said washing and combining it into the third volumetric flask, adding a further amount of water to the third volumetric flask until the volume reaches 10mL, and then shaking up the third volumetric flask to produce a fourth solution; centrifuging the fourth solution at 12000 rpm for 5-15 minutes to produce a centrifuged fourth solution, collecting a supernatant from the centrifuged fourth solution, measuring 1-3 mL of the supernatant accurately and placing it into a third plugged test tube of 10 mL, adding 1-3 mL 2,4-acetonitrile dinitrofluorobenzene solution having a concentration of 0.8% by weight and 1-3 mL sodium bicarbonate solution having a concentration of 0.5mol/L into the third tube to produce a fifth solution, heating up the fifth solution at 50-70°C in a water bath for 0.5-1.5 hours, taking out the fifth solution from the water bath and cooling it, and transferring it from the third tube into a fifth volumetric flask of 10 mL, washing the third tube with a phosphate buffer solution having a concentration of 0.2mol/L and a pH of 7.0 for several times, collecting the phosphate buffer solution after each of said washing and combining it into the fifth volumetric flask, adding a further amount of the phosphate buffer solution into the fifth volumetric flask until the volume reaches 10mL, shaking up, to provide a test sample solution;
accurately sucking 0.004-0.006 mL of the reference compound solution and 0.005-0.015 mL of the test sample solution, respectively injecting them into a liquid chromatogram instrument for determination, and determining Feculae Bombycis content in the pharmaceutical composition, counted according to pipecolic acid (C6H11NO2), whereby said pharmaceutical composition has a positive quality control when said Feculae Bombycis content is not less than 0.0001g.

9. The method of claim 8, wherein said step of identification is:
A Adding 0.5 g of the pharmaceutical composition in 5 mL anhydrous ethanol to produce a solution, refluxing the solution at 60°C in a water bath for 30 minutes and then letting the solution standing, taking a supernatant from the solution and evaporating the supernatant above a water bath until 1 mL is left, to provide a test sample solution;

furthermore, taking .beta.-sitosterol as a reference substance which is added with anhydrous ethanol to provide a reference substance solution to provide a reference substance solution having a concentration of-0.001 g/mL;
performing a thin layer chromatography test by sucking 0.002 mL of said test sample solution and 0.002 mL of said reference substance solution, spotting them on a same thin layer chromatography (TLC) silica gel plate and developing them by using a developing solvent composed of chloroform and acetone at a weight ratio of 19.5:0.5, then drying the silica gel plate, and spraying it with 10% phosphor-molybdic acid solution, followed by heating the silica gel plate at 105°C until a chromatogram is visualized; the chromatogram produced by the test sample solution shows the same color spots as that displayed by the reference substance solution;
B ldentification by high-performance liquid chromatography, using octadecylsilanized silica gel as a packing; using a mixture of methanol, ammonium acetate of 0.2mol/L and acetic acid at a weight ratio of 60:40:1 by weight as a mobile phase; detection wavelength is 250nm; theoretical plate number counted according to peak ammonium glycyrrhizinate is not less than 2000;
weighing 0.01 g mono-ammonium glycyrrhizinate accurately, dissolving it with said mobile phase in a volumetric flask to 50 mL and shaking up to provide a reference substance solution containing mono-ammonium glycyrrhizinate at 0.0002 g/mL, which is equivalent to 0.0001959 g/mL of glycyrrhizic acid;
taking 2 g of said pharmaceutical composition, putting it into a volumetric flask of 25 mL, adding 20 mL of said mobile phase, exposing the mixture to ultrasound treatment for 30 minutes with power 250W and frequency of 50KHz, then cooling the mixture, adding the mobile phase to the mixture until the volume reaches 25 mL, shaking up the mixture, followed by centrifuging the mixture, taking a supernatant from the centrifuged mixture for using as a test sample solution;
accurately sucking 0.01 mL of the reference substance solution and 0.01 mL of the test sample solution and injecting them into liquid chromatogram instrument, ensuring that the test sample solution should show a chromatographic peak having a same retention time as that of the reference substance solution; and said step of content determination is :

An applicability test of chromatogram condition and system using octadecylsilanized silica gel as a packing;
using a mixture of acetonitrile, dimethyl formamide and sodium acetate of 0.025mol/L at a weight ratio of 21:0.5:79 as a mobile phase;
detection wavelength is 390nm, the number of theoretical plates is not less than 2500 counted according to peak of pipecolic acid;
accurately weighing 0.01 g of pipecolic acid as reference compound, putting it into a first volumetric flask, adding water to dilute to 100 mL and shaking up to produce a first solution, measuring 1 mL of the first solution accurately and putting it into a first plugged test tube with a volume of 10 mL, adding 1 mL water, adding 2 mL 2,4-acetonitrile dinitrofluorobenzene solution of 0.8% by weight and 2 mL sodium bicarbonate solution having a concentration of 0.5mol/L to the tube to produce a second solution, heating the second solution at 60°C in a water bath for 1 hour, taking out the second solution in the tube from the water bath, cooling the second solution, and transferring the second solution into a second volumetric flask of 10 mL, washing the tube with phosphate buffer solution having a concentration of 0.2mol/L and a pH
value of 7.0 for 3 times, collecting the phosphate buffer solution after each of said washing and combining it into the second volumetric flask, adding a further amount of the phosphate buffer solution to the second volumetric flask until the volume reaches 10mL, and then shaking the second volumetric flask to provide a reference compound solution;
weighing 0.4 g of the pharmaceutical composition accurately, placing it in a second plugged test tube with a volume of 10 mL, adding 5 mL water in the tube and shaking it up fully to produce a third solution, heating up the tube containing the third solution at 60°C in a water bath for 30 minutes, taking out the tube and cooling the third solution, and transferring the third solution from the tube into a third volumetric flask of 10 mL. washing the tube with water for 3 times, collecting the water after each of' said washing and combining it into the third volumetric flask and adding a further amount of water to the third volumetric flask until the volume reaches 10mL, and then shaking up the third volumetric flask to produce a fourth solution;
centrifuging the fourth solution at 12000 rpm for 10 minutes to produce a centrifuged fourth solution, collecting a supernatant from the centrifuged fourth solution, measuring 2 mL of the supernatant accurately and placing it into a third plugged test tube of 10 mL, adding 2 mL of 2,4-acetonitrile dinitrofluorobenzene solution having a concentration of 0.8% by weight and 2 mL of sodium bicarbonate solution having a concentration of 0.5mol/L into the third tube to produce a fifth solution, heating up the fifth solution at 60°C in a water bath for 1 hour, taking out the fifth solution from the water bath and cooling it, and transferring it from the third plugged tube into a fifth volumetric flask of 10 mL, washing the third plugged tube with phosphate buffer solution having a concentration of 0.2mol/L and a pH of 7.0 for 3 times, collecting the phosphate buffer solution after each of said washing and combining it into the fifth volumetric flask, adding a further amount of the phosphate buffer solution into the fifth volumetric flask until the volume reaches 10mL, shaking up to provide a test sample solution;
accurately sucking 0.005 mL reference compound solution and 0.01 mL test sample solution, respectively injecting them into a liquid chromatogram instrument for determination, and determining Feculae Bombycis content in the pharmaceutical composition, counted according to pipecolic acid (C6H11NO2), whereby said pharmaceutical composition has a positive quality control when said Feculae Bombycis content is not less than 0.0002g.

10. Use of the pharmaceutical composition of any one of claims 1 to 5 in the preparation of a medicine for treating diabetes.

11. The use of claim 10, wherein said treating diabetes is to improve on the symptoms of polydipsia or polyphagia compared to a control group.

12. The use of claim 10, wherein said treating diabetes is to decrease blood glucose and serum fructosamine compared to a control group.

13. The use of claim 10, wherein said treating diabetes is to decrease serum cholesterol and triglyceride levels compared to a control group.

14. The use of claim 10, wherein said treating diabetes is to decrease serum NAG-ase activity and improve microvascular complications compared to a control group.

15. The use of claim 10, wherein said treating diabetes is to decrease sorbitol content in sciatic nerve and improve chronic neuropathy of diabetes compared to a control group.

16. The use of claim 10, wherein said treating diabetes is to increase GSH
content in red blood cell, and enhance body's antioxidant capacity compared to a control group.

17. The use of claim 10, wherein said treating diabetes is to lower kidney weight, body mass index, reduce serum creatinine levels or relieve glycogen deposition in renal tubular epithelial cells compared to a control group.

18. Use of said pharmaceutical composition of any one of claim 1 to 5 for treating diabetes.

19. The use of claim 18, wherein said treating diabetes is to improve on the symptoms of polydipsia or polyphagia compared to a control group.

20. The use of claim 18, wherein said treating diabetes is to decrease blood glucose and serum fructosamine compared to a control group.

21. The use of claim 18, wherein said treating diabetes is to decrease serum cholesterol and triglyceride levels compared to a control group.

22. The use of claim 18, wherein said treating diabetes is to decrease serum NAG-ase activity and improve microvascular complications compared to a control group.

23. The use of claim 18, wherein said treating diabetes is to decrease sorbitol content in sciatic nerve and improve chronic neuropathy of diabetes compared to a control group.

24. The use of claim 18, wherein said treating diabetes is to increase GSH
content in red blood cell, and enhance body's antioxidant capacity compared to a control group.

25. The use of claim 18, wherein said treating diabetes is to lower kidney weight, body mass index, reduce serum creatinine levels or relieve glycogen deposition in renal tubular epithelial cells compared to a control group.

26. The use of claim 18, wherein said treating diabetes is to inhibit .alpha.-glucosidase activity.