AU2007357043A1 - Pharmaceutical composition for regulating blood sugar and fat, preparation and use thereof - Google Patents

Description

Pharmaceutical composition for regulating blood sugar and fat, preparation and use thereof Technical Field The present invention relates to a pharmaceutical composition for regulating blood sugar and fat, in particular to a pharmaceutical composition prepared from Chinese herbs or extracts thereof as active ingredients, to a method for preparing the pharmaceutical composition, and to a use of the pharmaceutical composition for the treatment of diabetes mellitus and complications thereof. Background Art Diabetes mellitus, a common metabolic disease associated with endocrine, occurs all around the world, and exhibits a trend of increasing gradually. At present, the prevalence of diabetes mellitus in China is up to 1.5%. China has the second large population of diabetics in the world, and the population increases at a rate of doubling per 15 years. Among the population of diabetes are mainly suffered from diabetes mellitus type 1l. After occurrence of insulin resistance (i.e., a state that a normal dose of insulin produces a lower level of biological effect than normal effect), glucose tolerance is maintained or lightly impaired as long as pancreas could maintain sufficient insulin secretion to overcome the insulin resistance. Once failure of the function of beta-cell starts, the glucose tolerance deteriorates quickly, and diabetes mellitus develops. Insulin resistance is an important pathogenic factor of diabetes mellitus type 11 and the metabolic disorder of sugar is usually accompanied by a metabolic disorder of fat. Thus, it is the key point for the treatment of diabetes mellitus to alleviate insulin resistance, to control the metabolic disorder of sugar and fat, and to prevent the complications from occurrence. At present, most of the available traditional Chinese medicines function through a mechanism of supplementing Qi and nourishing Yin, and activating blood circulation to dissipate blood stasis, and have a significant effect of reducing blood sugar level, but show less potent effects of reducing blood fat and rectifying metabolic disorder of fat in diabetes mellitus in the treatment of diabetes mellitus

-I-

type 11. A treatment through a mechanism of supplementing Qi and nourishing Yin, and dissipating phlegm and dredging collaterals is seldom used in the treatment of diabetes mellitus type 11, but the treatment may have significant effects of rectifying the metabolic disorder of fat in diabetes mellitus type 11, while reduces the level of blood sugar. Contents of the Invention One object of the present invention is to provide a novel pharmaceutical composition for regulating blood sugar and fat. Another object of the present invention is to provide a method for manufacturing the pharmaceutical composition of the present invention. Still another object of the present invention is to provide a method for the treatment of diabetes mellitus and complications thereof using the pharmaceutical composition of the present invention, or a use of the pharmaceutical composition of the present invention in the manufacture of a medicament for the treatment of diabetes mellitus and complications thereof. The inventors of the present invention find after a long-term of extensive studies that a pharmaceutical composition for regulating blood sugar and fat, alleviating insulin resistance and effectively preventing and treating diabetes mellitus and complications thereof can be provided utilizing the mechanism of supplementing Qi and nourishing Yin, and dissipating phlegm and dredging collaterals. The objects as identified above are achieved based on the above finding. In one aspect, the present invention provides a pharmaceutical composition for regulating blood sugar and fat, composed of the following Chinese herbs or extracts thereof in the weight parts: Ligustrum lucidum (Fructus Ligustri Lucidui) 5-17, Mongolian mikvetch root (Radix Astragali Mongolici) 3-12, Chinese goldthread rhizome (Rhizoma Coptidis) 1-5, Lychee seed (Semen Litchi) 1-5, Kelp (Thailus Laminariae Japonicae) 1-6, and Common turmeric rhizome (Rhizoma Curcumae Longae) 1-6. Preferably, the present invention provides a pharmaceutical composition for regulating blood sugar and fat, composed of the following Chinese herbs or extracts thereof in the weight parts: Ligustrum lucidum 8-15, Mongolian mikvetch -2root 4-10, Chinese goldthread rhizome 2-3, Lychee seed 3-4, Kelp 3-5, and Common turmeric rhizome 3-5. Most preferably, the present invention provides a pharmaceutical composition for regulating blood sugar and fat, composed of the following Chinese herbs or extracts thereof in the weight parts: Ligustrum lucidum 8, Mongolian mikvetch root 4, Chinese goldthread rhizome 2, Lychee seed 4, Kelp 3, and Common turmeric rhizome 3. In the pharmaceutical composition according to the present invention, each of the Chinese herbs can be replaced by their solvent extracts, active fractions or active ingredients, wherein the extracts may be alcohol extracts, water extracts or volatile oils. For example, the pharmaceutical composition according to the present invention may comprise an alcohol extract of Ligustrum lucidum, an alcohol extract of Mongolian mikvetch root, an alcohol extract of Chinese goldthread rhizome, a volatile oil of Common turmeric rhizome, a water extract of Common turmeric rhizome, a water extract of Lychee seed and a water extract of Kelp in effective amounts for treating or ameliorating diabetes mellitus and complications thereof. The pharmaceutical composition according to the present invention may further comprise any pharmaceutically acceptable excipients, such as dextrin, preferably beta dextrin. In the pharmaceutical composition according to the present invention, the Chinese herbs can be replaced by or added/deleted according to the traditional Chinese medical theories, and the amounts of ingredients can be varied. For example, kelp may be absent or replaced by sea tangle (Laminaria japonica aresch) or seaweed (Thai/us Sargassi Pallidi); and Common turmeric rhizome may be absent or replaced by wenchow turmeric root tuber (Radix Curcumae Wenyujin) or blue turmeric rhizome (Rhizoma Curcumae Phaeocaulis). Therefore, the present invention also provides a pharmaceutical composition for regulating blood sugar and fat, composed of the following Chinese herbs or extracts thereof in the weight parts: Ligustrum lucidum (Fructus Ligustri Lucidui) 5-17, Mongolian mikvetch root (Radix Astragali Mongolici) 3-12, Chinese goldthread rhizome (Rhizoma Coptidis) 1-5, and Lychee seed (Semen Litchi) 1-5, and a pharmaceutically acceptable excipient. -3- The pharmaceutical composition according to the present invention may be formulated into a dosage form used commonly in the art together with any pharmaceutically acceptable excipients, such as tablets, granules or capsules. A mixture of the ingredients thereof may be used directly, or solvent extracts of the ingredients or mixture thereof may also used. The pharmaceutical composition according to the present invention exhibits spots with the same color as those of the controls at corresponding positions, when tested by using the thin layer chromatography described in the Part 1, Annex VIB of the Pharmacopoeia of the People's Republic of China, 2005 ed. and using oleanolic acid, berberine hydrochloride, Astragaloside IV and protocatechuic acid as controls, respectively. The specific identification is shown as follow. (1) 2g of the pharmaceutical composition of the present invention is weighed, 5ml anhydrous ethanol is added, the obtained mixture is treated with ultrasonic waves for 25 min, cooled and filtered, and the filtrate is used as a test sample solution. Oleanolic acid as control is dissolved in anhydrous ethanol to form a 0.5 mg/ml solution as a control solution. According to the thin layer chromatography (Annex VIB), the test sample solution 5pl and the control solution 10pl are spotted on one silica gel G thin layer plate with sodium carboxymethylcellulose as the adhesive, and petroleum ether-acetone-methanol (8:2:0.3) is used as developing solvent. After development is finished, the plate is taken out, open-air dried, sprayed with 10% sulfuric acid ethanol solution, and heated at 80 *C until spots are colored clearly. The chromatography of the test sample exhibits spots with the same color as those of the control at the corresponding positions of the control chromatography. (2) 0.5g of the pharmaceutical composition of the present invention is weighed, 5ml methanol is added, the obtained mixture is treated with ultrasonic waves for 30 min, cooled and filtered, and the filtrate is supplemented with methanol to 5ml and used as a test sample solution. Berberine hydrochloride as control is dissolved in methanol to form a 0.1mg/mi solution as a control solution. According to the thin layer chromatography (Annex VIB), the test sample solution 2pl and the control solution 2pl are spotted on one silica gel G thin layer plate with sodium carboxymethylcellulose as the adhesive, and benzene-ethyl -4acetate-isopropanol-methanol-water (6:3:1.5:1.5:0.3) is used as developing solvent. After the development is finished in an ammonia gas-saturated developing tank, the plate is taken out, open-air dried, and visually inspected under UV light (365nm). The chromatography of the test sample exhibits fluorescent spots with the same color as those of the control at the corresponding positions of the control chromatography. (3) 6g of the pharmaceutical composition of the present invention is weighed, 20ml solution of 2% NaOH in methanol is added, the obtained mixture is heated under reflux for 40 min, cooled and filtered, the filtrate is evaporated to dry, the residue is dissolved in 30ml water, and extracted with water-saturated n-butanol for three times (30ml, 20ml and 20ml). The n-butanol layers are combined, washed twice with water of 20ml each, and after water layers are discarded, washed 1% potassium dihydrogen phosphate solution once. The water layer is discarded, the n-butanol layer is evaporated to dry and the obtained residue is dissolved in 5ml methanol to form a test sample solution. Astragaloside IV as control is dissolved in methanol to form a 0.3mg/ml solution as a control solution. According to the thin layer chromatography (Annex VIB), the test sample solution 10-15pl and the control solution 10pl are spotted on one silica gel G thin layer plate with sodium carboxymethylcellulose as the adhesive, the lower layer solution of chloroform-methanol-water (20:7:2) is used as developing solvent. After the development is finished, the plate is taken out, open-air dried, sprayed with 10% sulfuric acid ethanol solution, and heated at 80 0C until the sports are clear in their colors. The chromatography of the test sample exhibits spots with the same color as those of the control at the corresponding positions of the control chromatography. (4) 3g of the pharmaceutical composition of the present invention is weighed, 50ml water is added, the obtained mixture is heated under reflux for 1 h, cooled and filtered, the filtrate is adjusted with 10% HCI until pH=2, the acidized solution is extracted with ethyl ether for three times of 15ml each, ethyl ether layers are combined and ethyl ether is recovered. The obtained residue is dissolved in 1ml methanol to form a test sample solution. Protocatechuic acid as control is dissolved in methanol to form a 0.2mg/ml solution as a control solution. According to the thin -5layer chromatography (Annex VIB), the test sample solution 10pl and the control solution 8pl are spotted on one silica G thin layer plate with sodium carboxymethylcellulose as the adhesive, toluene-ethyl acetate-formic acid (12:6:1) is used as a developing solvent. After the development is finished, the plate is taken out, open-air dried, and visually inspected under UV light (254nm). The chromatography of the test sample exhibits spots with the same color as those of the control at the corresponding positions of the control chromatography. The pharmaceutical composition according to the present invention comprises oleanolic acid and ursolic acid in amounts of not less than 4.0mg and 1.0mg, respectively, when tested by using the high performance liquid chromatography in the Part 1, Annex VI D of the Pharmacopoeia of the People's Republic of China, 2005 ed. and using oleanolic acid and ursolic acid as controls, respectively. The specific detection is described as follow. Octadecylsilane bonded silica gel is used as packing; methanol-0.05% phosphoric acid water (90:10) is used as mobile phase; and detection wavelength is 215nm. Theoretical plate number is not less than 3,000 as calculated using oleanolic acid. Appropriate amount of Oleanolic acid and ursolic acid as controls are precisely weighed and dissolved with acetonitrile to form 0.20mg/ml oleanolic acid solution and 0.15mg/ml ursolic acid solution as control solutions, respectively. The pharmaceutical composition according to the present invention under content uniformity is comminuted, 0.8g thereof is precisely weighed, and 20ml methanol is added. The resulting mixture is heated under reflux for 45 min, cooled, and transferred in a 50ml volumetric flask. A small amount of methanol is used to wash the container and residue, and the washes are transferred in the same volumetric flask. The solution in the volumetric flask is agitated to be homogeneous and filtered by using microporous filter membrane (0.45pm), and the filtrate is used as a test sample solution. The control solutions 15pl each and the test sample solution lOpI are injected into the liquid phase chromatographic instrument and detected, showing that the pharmaceutical composition of the present invention comprises oleanolic acid (C 30

H

4 8 0 3 ) not less than 4.0mg. In another aspect, the present invention provides a method for preparing the pharmaceutical composition of the present invention, the method comprising the -6following steps: Extracting Ligustrum lucidum with ethanol by heating under reflux for several times, combining ethanol extract solutions, recovering ethanol, and drying the extract for standby; Extracting Common turmeric rhizome by water steam distillation, collecting the volatile oil, the water solution and the residue for standby; Including the obtained volatile oil with cyclodextrin and water, stirring, freezing, filtering, cryodrying the obtained inclusion, pulverizing for standby; Decocting lychee seed and kelp with water for several times, combining decocting solutions, filtering, combining the filtrate and the above water solution of Common turmeric rhizome, concentrating, adding alcohol, freezing, filtering to obtain a water decocting-alcohol settling solution for standby; and Extracting Chinese goldthread rhizome, Mongolian milkvetch root and the above residue of Common turmeric rhizome with alcohol by heating under reflux for several times, combining alcohol extract solutions, combining with the above water decocting-alcohol settling solution, recovering alcohol, drying, mixing homogeneously with the above extract of Ligustrum lucidum and the cyclodextrin inclusion of Common turmeric rhizome, mixing with pharmaceutically acceptable excipients to form desired dosage forms, if necessary. The steps and conditions of the preparation method according to the present invention can be optimized according to the following experimental results. 1. Optimization of conditions for the extraction and inclusion of Common turmeric rhizome volatile oil 1.1 Effects of comminution degree on extract quantity of volatile oil of Common turmeric rhizome, and determination of time for extracting the volatile oil Two parts of Common turmeric rhizome (100g for each part) were weighed, the specifications of the two parts were respectively decoction pieces, and 10 mesh. Water in 10 times amount is added, respectively, and volatile oils were extracted by water steam distillation. The results are shown in Table 1. -7- Table 1. Investigation of time for extracting volatile oil of Common turmeric Rhizome and comminution degree Extraction time (h) 1 2 3 4 5 6 7 8 9 Yield, decoction 0.4 0.8 1.0 1.2 1.4 1.5 1.6 1.95 2.0 pieces (ml) Yield, 10 mesh (%) 1.4 2.2 3.1 3.6 3.8 4.1 4.4 4.6 4.7 The above results show that the comminution degree significantly influences the yield of volatile oil of Common turmeric rhizome. Thus, a suitable comminution degree is needed for extracting volatile oil of Common turmeric rhizome. In order to ensure no coking occurs in production, the comminution degree of Common turmeric rhizome is chosen as 10 mesh. The yield of volatile oil of 10 mesh Common turmeric rhizome was 4.7ml at 9 h of extraction, 4.6ml at 8 h of extraction, which is to say that 97% of the yield was achieved for 8 h. Therefore, the time for extracting volatile oil of Common turmeric rhizome is determined as 8 h. 1.2 Effects of soaking on extraction quantity of the volatile oil Two parts of Common turmeric rhizome (100g per part) were weighed, in which one part was directly extracted without soaking, while the other part is soaked for 2 h before extraction. The results are shown in Table 2. Table 2. Effects of soaking on the extraction of volatile oil of Common turmeric rhizome Extraction time (h) 1 2 3 4 5 6 7 8 Soaking (ml) 0.4 1.4 1.9 2.2 2.6 2.8 3.2 3.4 No-soaking (ml) 1.4 2.2 3.1 3.6 3.8 4.1 4.4 4.6 The above results show that the effects of soaking on the extraction quantity of volatile oil of Common turmeric rhizome were not significant. Therefore, no socking is needed for extracting the volatile oil of 10 mesh Common turmeric rhizome. 2. Effects of water amount on the extraction of volatile oil of Common turmeric -8rhizome Table 3. Effects of water amount on the extraction of volatile oil of Common turmeric rhizome Extraction time (h) 1 2 3 4 5 6 7 8 8 times amount of water 1.4 1.9 2.3 2.7 3.3 3.6 3.9 4.1 10 times amount of water 1.4 2.2 3.1 3.6 3.8 4.1 4.4 4.6 12 times amount of water 1.3 2.3 2.8 3.3 3.7 3.9 4.3 4.6 The above results show that the yields of volatile oil of Common turmeric rhizome were higher with 10 times amount of water and 12 times amount of water used in the extraction than that with 8 times amount of water, while the yields for 10 times and 12 times of water were close. Therefore, 10 times amount of water is chosen. In sum, the optimized conditions for extracting volatile oil of Common turmeric rhizome are: 10 mesh Common turmeric rhizome and 10 times amount of water are used, the volatile oil of Common turmeric rhizome is extracted by water steam distillation, and the time for extracting volatile oil of Common turmeric rhizome is 8 h. 3. Optimizing the process for inclusion of volatile oil of Common turmeric rhizome The inclusion of volatile oil of Common turmeric rhizome is carried out by using 6 times amount of beta cyclodextrin and 60 times amount of water, stirring at 70 0 C for 1 h, refrigerating overnight, filtering and drying the inclusion at a low temperature (40 0 C), pulverizing to obtain a fine powder for standby. 4. Optimization of conditions for extracting Ligustrum lucidum with alcohol 20g Ligustrum lucidum was weighed as one sample, and there were 9 samples in total. The alcohol concentration, the multiple of alcohol amount and the number of times of alcohol extraction were used as three factors investigated in an orthogonal experiment. Each of the factors had three levels (see Table 4). The extraction was conducted for 2 h for each time. The experiment was performed in -9accordance with L 9 (3 4 ) orthogonal layout. The oleanolic acid content and ursolic acid content in Ligustrum lucidum were used as the observed indexes. The design of factors and levels are shown in Table 4. The orthogonal experiment and results thereof are shown in Table 5, and the statistical treatment of the results in Table 5 is shown in the variance analysis tables (see Table 6 and Table 7). Table 4. Levels of alcohol extraction factors A (Alcohol B (Multiple of alcohol C (times of alcohol concentration, %) amount) extraction) 1 55 4 1 2 75 5 2 3 95 6 3 Table 5. Scheme and results of the orthogonal experiment Oleanolic acid Ursolic acid No. A B C D content in extract content in extract (mg/g) (mg/g) 1 1 1 1 1 2.24 0.42 2 1 2 2 2 4.83 1.02 3 1 3 3 3 7.28 1.90 4 2 1 2 3 7.65 2.17 5 2 2 3 1 8.13 2.19 6 2 3 1 2 6.34 1.97 7 3 1 3 2 6.94 2.12 8 3 2 1 3 6.26 1.99 9 3 3 2 1 7.26 2.58 Oleanolic acid: KI 14.35 16.83 14.84 17.63 K2 22.12 19.22 19.74 18.11 E=56.93 K3 20.46 20.88 22.35 21.19 CT=360.1138777 S 11.1623 2.7633556 9.6913556 2.4878223 Ursolic acid: K1 3.34 4.71 4.38 5.19 K2 6.33 5.2 5.77 5.11 Z=16.36 K3 6.69 6.45 6.21 6.06 CT=29.73884444 S 2.25468889 0.53668889 0.60828889 0.18508889 -10- Table 6. Variance analysis table for oleanolicacid acid Source Sum of square Degree of Mean sum F-ratio Significance freedom of square A 11.1623 2 5.58115 4.4868 B 2.7633556 2 1.3816778 1.1108 C 9.6913556 2 4.8456778 3.8955 Error=D 2.4878223 2 1.24391115

F

1 - 05(2,2)=19 F 10 o01(2,2)=99 Table 7. Variance analysis table for ursolic acid Source Sum of square Degree of Mean sum F-ratio Significance freedom of square A 2.25468889 2 1.127344445 12.1817 B 0.53668889 2 0.268344445 2.8996 C 0.60828889 2 0.304144445 3.2865 Error=D 0.18508889 2 0.092544445

F

1

-

0 05(2.2)=19 F1 -. 01( 2

,

2 )=99 The above variance analyses show that all three factors did not have significant difference. In accordance with the range analysis, the extraction conditions for oleanolic acid are A 2

B

3

C

3 , and the extraction conditions for ursolic acid are A 3

B

3

C

3 . Since the alcohol concentration has no significant influence on the extraction, A 2 is more favorable for the extraction of oleanolic acid. Thus, A 2

B

3

C

3 are finally chosen, i.e., the extraction conditions for Ligustrum lucidum are: extracting 3 times, each for 2 h, by using 75% ethanol in 6 times amount. 5. Optimization of alcohol extraction conditions for Chinese goldthread rhizome and Mongolian milkvetch root 5.1. Orthogonal experiment for Chinese goldthread rhizome and Mongolian milkvetch root 15g Chinese goldthread rhizome and 30g Mongolian milkvetch root were weighed as one sample, and there were 9 samples in total. The alcohol -11concentration, the multiple of alcohol amount and the number of times of alcohol extraction were used as three factors investigated in an orthogonal experiment. Each of the factors had three levels (see Table 4). The extraction was conducted for 2 h for each time. The experiment was performed in accordance with L 9 (3 4 ) orthogonal layout. The berberine hydrochloride content in Chinese goldthread rhizome was used as the observed index. The design of factors and levels are shown in Table 8. The orthogonal experiment and results thereof are shown in Table 9, and the statistical treatment of the results in Table 9 is shown in the variance analysis table (see Table 10). Table 8. Levels of alcohol extraction factors A (Alcohol B (Multiple of alcohol C (Number of times of Level concentration, %) amount) alcohol extraction) 1 30 4 1 2 50 5 2 3 70 6 3 Table 9. Scheme and results of the orthogonal experiment No. A B C D Berberine hydrochloride content in extract (mg/g) 1 1 1 1 1 10.86 2 1 2 2 2 17.14 3 1 3 3 3 19.39 4 2 1 2 3 18.58 5 2 2 3 1 21.38 6 2 3 1 2 14.04 7 3 1 3 2 20.71 8 3 2 1 3 13.53 9 3 3 2 1 19.55 K1 47.39 50.15 38.43 51.79 K2 54.00 52.05 55.27 51.89 E=155.18 K3 53.79 52.98 61.48 51.50 CT=2675.648044 S 9.410689 1.387089 94.827889 0.027356 -12- Table 10. Variance analysis table for berberine hydrochloride Sum of Degree of Mean sum of Source F-ratio Significance square freedom square A 9.410689 2 4.7053445 344.0082 ** B 1.387089 2 0.6935445 50.7051 * C 94.827889 2 47.4139445 3466.4384 ** Erro=D 0.027356 2 0.013678

F

1 -o.1(22)=l 9 F 1 -. o5(2,2)=99 The above variance analysis shows that all three factors had significant differences, A 2 is chosen for factor A, B 3 is chosen for factor B and C3 is chosen for factor C. Therefore, A 2

B

3

C

3 were chosen as the optimal extraction conditions for berberine hydrochloride, i.e., extracting 3 times, each for 2 h, by using 50% ethanol in 6 times amount. 6. Verification of the orthogonal experiment for Chinese goldthread rhizome and Mongolian milkvetch root In order to verify the above experimental conditions, Chinese goldthread rhizome and Mongolian milkvetch root were weighed, and extract samples were prepared in accordance with the abovementioned optimal conditions. The berberine hydrochloride contents of the extracts were measured and shown in Table 11. Table 11. Verification of orthogonal experiment of berberine hydrochloride Berberine hydrochloride Samples Quantity of sample content (mg/g) First sample Chinese goldthread rhizome 15g, 21.9882 Mongolian milkvetch root 30g Second sample Chinese goldthread rhizome 15g, 22.2236 Mongolian milkvetch root 30g 7. Optimization of conditions for the decoction with water -13- 24g lychee seed and 18g kelp were weighed as one sample, and there were 9 samples in total. The multiple of water amount, decoction time, and the number of times of decoction were used as three factors observed in an orthogonal experiment. Each of the factors had three levels. The experiment was performed in accordance with L 9 (34) orthogonal layout. The protocatechuic acid content in lychee seed was used as the observed index. The design of factors and levels are shown in Table 12. The orthogonal experiment and results thereof are shown in Table 13, and the statistical treatment of the results in Table 13 is shown in the variance analysis table (see Table 14). Table 12. Factors and levels Level A (multiple of B (Decoction time) (Number of times water amount) of decoction) 1 6 0.5 1 2 8 1.0 2 3 10 1.5 3 Table 13. Scheme and results of the orthogonal experiment No. A B C D Protocatechuric acid content in extract (mg/g) 1 1 1 1 1 2 1 2 2 2 3 1 3 3 3 4 2 1 2 3 5 2 2 3 1 6 2 3 1 2 7 3 1 3 2 8 3 2 1 3 9 3 3 2 1 K1 10.0855 14.619 6.1112 12.4708 k2 13.2996 12.0689 16.009 13.1835 7=39.3775 k3 15.9924 12.6896 17.2573 13.7232 CT=172.2875007 S 5.8303421 1.1789848 24.8622492 0.26308030 -14- Table 14. Variance analysis table for protocatechuic acid Degree of Mean sum of Source Sum of square F-ratio Significance freedom square A 5.8303421 2 2.91517105 22.16183416 * B 1.1789848 2 0.5894924 4.481463644 C 24.8622492 2 12.4311246 94.50441253 * Error=D 0.26308030 2 0.13154015 Fl-0.05(2,2)=19 F1-0.01(2,2)=99 The above variance analysis shows that factors A and C had significant differences and factor B had no significant difference. Thus, A 3 is chosen for factor A, B 1 is chosen for factor B and C3 is chosen for factor C, i.e., A 3

B

1

C

3 . The optimal conditions for the decoction of lychee seed and kelp are: extracting 3 times, each for 0.5 h, by using water in 10 times amount. The pharmaceutical composition of the present invention was studied in diabetes mellitus type il models in rat. Insulin resistance was successfully induced in rats by feeding rats with a high-fat and high-sugar diet up to 1 month when hyperinsulinism accompanied by increased level of bolod cholesterol and sugur. The diabetes mellitus type 11 rat models were established by intraperitoneal injection of a small dosage of STZ. After oral gavage for consecutive two months, blood sugar level of rats in the large dosage of the present composition group decreased significantly (P<0.001, in comparison with the model group), and the rate of blood sugar decrease was 31.23%. Blood fat level of rats in the large and middle dosage groups decreased significantly (P<0.05-0.001, in comparison with the model group), respectively. Thus, the pharmaceutical composition of the present invention exhibits effects of reducing sugar and fat in rats having diabetes mellitus type 1l. Quickly onset diabetes mellitus (diabetes mellitus type 1) rat models were established by the injection of STZ. The results showed that after oral gavage for -15consecutive two months, the blood sugar levels of rats in all dosage groups decreased significantly (P<0.05, in comparison with the model group). As compared with the model group, the glycosylated hemoglobin and the percentage of glycosylated hemoglobin decreased significantly (P<0.05-0.01), and the glucagon also decreased (P<0.05) in the large dosage group. Although insulin level exhibited a trend of increase and food consumption decreased in some extent, they are not statistically significant. Thus, the pharmaceutical composition of the present invention exhibits effects of reducing blood sugar level in rats having quickly onset diabetes mellitus. Thus, the present invention further provides a method for the treatment of diabetes mellitus and complications thereof by using a pharmaceutical composition of the present invention. Alternatively, use of a pharmaceutical composition of the present invention in the manufacture of a medicament for the treatment of diabetes mellitus and complications thereof is provided. Concrete Modes for Carrying Out the Invention The Chinese herbs used in the following experiments were purchased in Anguo Trading Market of Chinese herbs, Hebei Province, and they all met the relevant requirements specified in the Pharmacopoeia of the People's Republic of China, Part one , 2005 Edition. Dextrin was purchased from Langfang Starch Plant, Hebei Province, and had a batch No. 200308202 and an approved number: (1998) No. 090127. Beta-cyclodextrin was purchased from Fine Chemicals Experimental Plant of Nankai University, and had a batch No. 20000804. All other reagents were obtained commercially from market. The present invention is further illustrated but not restricted by the following examples. Example 1: Preparation of a pharmaceutical composition of the present invention (in form of granules) 1. Formulation Ligustrum lucidum 9g; Mongolian milkvetch root 6g; Chinese goldthread -16rhizome 4.5g; Lychee seed 3g; Kelp 3g; Common turmeric rhizome3g; and Dextrin in appropriate amount. 2. Process of preparation Ligustrum lucidum was heated with 75% ethanol in 6 times amount under refluxing and extracted for 3 times, each for 2 h. All ethanol extracts were combined for standby. Chinese goldthread rhizome and Mongolian mikivetch root were heated with 50% ethanol in 6 times amount under refluxing and extracted for 3 times, each for 2 h. All ethanol extracts were combined for standby. Common turmeric rhizome was broken into fine pieces, water in 10 times amount was added, extraction was performed by water steam distillation for 8 h, and volatile oil, water solution and residue were collected for standby. The inclusion of volatile oil was obtained by using beta-cyclodextrin in 8 times amount and water in 80 times amount, stirring at 50 0 C for 1 h, storing overnight under cold condition and filtering. The inclusion was dried at a low temperature (40*C), and comminuted to obtain a fine powder for standby. Lychee seed and kelp were extracted with water in 10 times amount for 3 times, each for 0.5h, in which the above residue of Common turmeric rhizome was combined and decocted in the third extraction, and all extracts were combined and filtered. The filtrate was combined with the above water solution, and the combined solution was concentrated until the relative density was 1.10-1.15 (measured at 50*C); then ethanol was added to reach an alcohol content of 60%, and the resultant solution was stored under cold condition overnight and filtered. The filtrate and the above solutions such as the extract of Ligustrum lucidum, etc. were respectively subjected to ethanol recovery until their relative densities were 1.15-1.20 (measured at 50 0 C), dried, mixed with the fine powder of beta-cyclodextrin inclusion of the volatile oil and an appropriate amount of dextrin, processed to form granules, and dried to form granules (1 Og). Example 2: Preparation of the pharmaceutical composition of the present invention(tablets) 1. Formulation Ligustrum lucidum 22.5g; Mongolian milkvetch root 15g; Chinese goldthread rhizome 4.5g; Lychee seed 6g; Kelp 7.5g; Common turmeric rhizome 7.5g; and -17- Dextrin in appropriate amount. 2. Process of preparation Ligustrum lucidum was heated with 75% ethanol in 6 times amount under refluxing and extracted for 3 times, each for 2 h. All ethanol extracts were combined, subjected to ethanol recovery, and dried for standby. Common turmeric rhizome was broken into fine pieces, water in 10 times amount was added, extraction was performed by water steam distillation for 8 h, volatile oil and water solution were collected for standby, and residue was dried for standby. The inclusion of volatile oil was obtained by using beta-cyclodextrin in 8 times amount and water in 80 times amount, stirring at 500C for 1 h, storing overnight under cold condition and filtering. The inclusion was dried at a low temperature (40 0 C), and comminuted to obtain a fine powder for standby. Lychee seed and kelp were decocted with water in 10 times amount for 3 times, each for 0.5h, the extracts were combined and filtered, the filtrate was combined with the above water solution of Common turmeric rhizome, and the combined solution was concentrated until the relative density was 1.10-1.15 (measured at 500C); then ethanol was added to reach an alcohol content of 60%, and the resultant solution was stored under cold condition overnight and filtered to obtain a filtrate for standby. Chinese goldthread rhizome, Mongolian miklvetch root and the residue of Common turmeric rhizome were heated with 50% ethanol in 6 times amount under refluxing and extracted for 3 times, each for 2 h. All ethanol extracts were combined, and then combined with the water decocting-alcohol settling solution. The combined solution was subjected to ethanol recovery until its relative density was 1.15-1.20 (measured at 500C), dried, mixed homogeneously with the extract of Ligustrum lucidum, the fine powder of beta-cyclodextrin inclusion and an appropriate amount of dextrin, processed to form granules, dried to form tablets (0.72-0.75g/tablet, 9 tablets in total). Example 3: Preparation of the pharmaceutical composition of the present invention(capsules) 1. Formulation Ligustrum lucidum 25.5g; Mongolian milkvetch root 18g; Chinese goldthread rhizome 7.5g; Lychee seed 7.5g; Kelp 9g; Common turmeric rhizome 9g; and -18- Dextrin in appropriate amount 2. Process of preparation Ligustrum lucidum was heated with 75% ethanol in 6 times amount under refluxing and extracted for 3 times, each for 2 h. All ethanol extracts were combined, subjected to ethanol recovery, and dried for standby. Common turmeric rhizome was broken into fine pieces, water in 10 times amount was added, extraction was performed by water steam distillation for 8 h, volatile oil and water solution were collected for standby, and residue was dried for standby. The inclusion of volatile oil was obtained by using beta-cyclodextrin in 8 times amount and water in 80 times amount, stirring at 50*C for 1 h, storing overnight under cold condition and filtering. The inclusion was dried at a low temperature (40*C), and comminuted to obtain a fine powder for standby. Lychee seed and kelp were decocted with water in 10 times amount for 3 times, each for 0.5h, the extracts were combined and filtered, the filtrate was combined with the above water solution of Common turmeric rhizome, and the combined solution was concentrated until the relative density was 1.10-1.15 (measured at 50*C); then ethanol was added to reach an alcohol content of 60%, and the resultant solution was stored under cold condition overnight and filtered to obtain a filtrate for standby. Chinese goldthread rhizome, Mongolian miklvetch root and the residue of Common turmeric rhizome were heated with 50% ethanol in 6 times amount under refluxing and extracted for 3 times, each for 2 h. All ethanol extracts were combined, and then combined with the water decocting-alcohol settling solution. The combined solution was subjected to ethanol recovery until its relative density was 1.15-1.20 (measured at 500C), dried, mixed homogeneously with the extract of Ligustrum lucidum, the fine powder of beta-cyclodextrin inclusion and an appropriate amount of dextrin, processed to form granules, dried, and capsulated to form capsules (0.43g/capsule, 15 capsules in total). Example 4: Preparation of the pharmaceutical composition of the present invention (extract paste) 1. Formulation Ligustrum lucidum 12g; Mongolian milkvetch root 6g; Chinese goldthread -19rhizome 3g; Lychee seed 6g; Kelp 4.5g; Common turmeric rhizome4.5g; and Dextrin in appropriate amount. 2. Process of preparation Ligustrum lucidum was heated with 75% ethanol in 6 times amount under refluxing and extracted for 3 times, each for 2 h. All ethanol extracts were combined, subjected to ethanol recovery, and dried for standby. Common turmeric rhizome was broken into fine pieces, water in 10 times amount was added, extraction was performed by water steam distillation for 8 h, volatile oil and water solution were collected for standby, and residue was dried for standby. The inclusion of volatile oil was obtained by using beta-cyclodextrin in 8 times amount and water in 80 times amount, stirring at 500C for 1 h, storing overnight under cold condition and filtering. The inclusion was dried at a low temperature (400C), and comminuted to obtain a fine powder for standby. Lychee seed and kelp were decocted with water in 10 times amount for 3 times, each for 0.5h, the extracts were combined and filtered, the filtrate was combined with the above water solution of Common turmeric rhizome, and the combined solution was concentrated until the relative density was 1.10-1.15 (measured at 500C); then ethanol was added to reach an alcohol content of 60%, and the resultant solution was stored under cold condition overnight and filtered to obtain a filtrate for standby. Chinese goldthread rhizome, Mongolian miklvetch root and the residue of Common turmeric rhizome were heated with 50% ethanol in 6 times amount under refluxing and extracted for 3 times, each for 2 h. All ethanol extracts were combined, and then combined with the water decocting-alcohol settling solution. The combined solution was subjected to ethanol recovery until its relative density was 1.15-1.20 (measured at 500C), mixed homogeneously with the extract of Ligustrum lucidum, the fine powder of beta-cyclodextrin inclusion to form the extract paste. Example 5: Experimental study of the pharmaceutical composition of the present invention in rats having diabetes mellitus type 11 1. Experimental materials 1.1. Animals Wistar grade Il rats, half male and half female, with body weight 180-200g -20were provided by the Institute of Laboratory Animals, Chinese Academy of Medical Sciences, License No. SCXK (Beijing) 2000-0006. The rats were bred in the Center of Laboratory Animals of Xiyuan Hospital of Chinese Academy of Medical Sciences, Second grade animal breading facility, Certificate No. SYXK (Beijing) 2003-0008. Room temperature: 22-25 0 C, relative humidity: 45-60%. 1.2. Diets Common diets were provided by Beijing Ke Ao Xie Li Diets Company, product license No.: Beijing (formulation) 238, practiced standard: GB14924, 3-2001. Essential nutrients: crude protein 218%, crude fibers 55%, crude ash 58%, calcium 0.8-1.8%, phosphorus 0.6-1.2%, lysine 1.35%, sodium chloride 0.5%, water 510%, various vitamins and trace elements. High-fat diets were prepared by the Center of Laboratory Animals, Chinese Academy of Medical Sciences. Formula: pork fat 10%, sucrose 10%, cholesterol 2.5%, sodium cholate 1%, common diet 76.5%. 1.3. Drugs The pharmaceutical composition of the present invention: extract paste, 3.37g crude herb per gram of paste, batch No. 050401, prepared in accordance with the method of Example 4. Metformin hydrochloride tablets: 0.25g/tablet, batch No. 041226, Beijing Li Ling Heng Tai Pharmaceutical Company, GUOYAOZHUNZI H11021560. Jin Qi Jiang Tang tablets: 0.42g/tablet, batch No. 0503746, produced by Long Shun Rong Pharmaceutical Factory of Tianjin Zhong Xin Pharmaceutical Group Company, GUOYAOZHUNZI Z10920027. 1.4. Reagents Citric acid and sodium citrate: Beijing Chemical Plant, batch No. 960904. Streptozotocin (STZ): produced by Sigma Company, batch No. S0130, and subpackaged by Beijing Tian Lai Biomedicine Science Company. Blood sugar test paper: produced by Roche Company, batch No. 22898731, and subpackaged by Roche-Diagnostics (Shanghai) Company, Ltd. Radio-immunity kits (insulin, tumor necrosis factor, interleukin-6): Tianjin Jiu Ding Medical Bioengineering Company, Ltd., batch No. 200510. Biochemical kits (total cholesterol, triglycerides): Zhong -21- Sheng Bei Kong Biological Science Corporation, batch No. 050331. 50% Glucose injection: produced by Beijing Shuang He Pharmaceutical Corporation, batch No. 0502142. 2. Experimental methods Common diet group and high-fat, high-sugar diet group were designed. Insulin resistance was induced in rats after being fed with the high-fat and high-sugar diet for one month, and then hyperglycemia was induced by intraperitoneal injection of 25mg/kg STZ twice. The rats with a fasting blood sugar of greater than 16.7mmol/1 measured after 72 h were deemed as successful models, and were divided into the following groups: (1) blank control group (common diet + intraperitoneal injection of 0.1M citric acid buffer); (2) model group; (3) positive metformin group (0.2g/kg); (4) positive Jin Qi Jiang Tang tablet group (2g/kg); (5) the pharmaceutical composition of the present invention, large dosage group (8g crude herb/kg); (6) the pharmaceutical composition of the present invention, middle dosage group (4g crude herb/kg); (7) the pharmaceutical composition of the present invention, small dosage group (2g crude herb/kg), in which the rats of groups (2) to (7) were fed with the high-fat high-sugar diet and intraperitoneally injected with STZ-0.1M citric acid buffer. Except the control group and the model group, the rats of other groups were subjected to oral gavage and fed with the high-fat and high-sugar diet for one month. Body weight and food consumption were measured every week, and blood sugar was measured every other week. After consecutive tow month of administration, blood samples were obtained from the abdominal aorta of the anesthetized rats, and measured to determine indexes, such as blood sugar, blood fat, insulin, tumor necrosis factor, interleukin-6, etc. Pancreas and liver were used to make pathological slices, and heart, liver and kidney were weighed to calculate organ index: organ weight/body weight X 100 (g/1 00g). The results were subjected to statistical treatment (t-test). 3. Results 3.1. Effects of the pharmaceutical composition of the present invention on blood sugar of rats having diabetes mellitus type 11 The rats of the groups fed with the high-fat high-sugar diet were subjected to intraperitoneal injection of STZ, then their fasting blood sugar levels were -22measured after 72 h, the blood sugar increased significantly (P<0.001) in the groups as compared to the control group before the administration of drugs, and the average blood sugar level is about 25-26mmol/l, which means the models were successfully established. The rats of the groups were subjected to oral gavage of drugs for two consecutive months. As compared to the model group, the blood sugar of rats of the large dosage group for the pharmaceutical composition of the present invention decreased at the 2 nd, 4 th and 6 t" week (P<0.05-0.01), and significantly decreased at the 8 'h week (P<0.001). The rate of blood sugar decrease was 31.23%. The results are shown in Table 15. Table 15. Effects of the pharmaceutical composition of the present invention on Blood sugar of rats having diabetes mellitus type il (mmol/l, -±s) Group Dose n Before 2 n" week after 4 ' week after 6 h week after 8 ' week after (g/kg) administration administration administration administration administration Control group - 12 5.62±0.39 5.63±0.42 5.08±0.32 5.87±0.49 4.22±0.61 Model group - 11 26.43±3.01*** 32.19±1.06 *** 33.00±00 *** 29.79±3.25 25.24±3.47 *** Metformin group 0.2 12 25.34±3.40*** 23.33±2.41### 28.04±4.16### 25.21±3.21 ## 17.79±2.65 ### Jin Qi Jiang Tang group 2 8 25.54±4.21** 29.58±2.46## 31.71±2.61 26.65±2.70 # 21.16±3.25# Large dosage group 8 11 26.42±3.16*** 28.73±3.58## 30.81±2.62## 26.67±3.54# 17.35±3.13### Middle dosage group 4 10 26.59±3.79*** 28.95±2.73## 32.39±1.21 25.38±2.83## 26.05±3.85 Small dosage group 2 11 26.24±3.81*** 30.83±2.38 32.83±0.39 28.94±2.79 27.05±3.55 As compared with the control group: * P<0.01, *** P<0.001; As compared with the model group: # P<0.05, ## P<0.01, ### P<0.001 3.2. Effects of the pharmaceutical composition of the present invention on blood fat of rats having diabetes mellitus type i After the rats were subjected to the oral gavage of drugs for consecutive two months, as compared to the model group, the large dosage group for the pharmaceutical composition of the present invention exhibited a significant decrease in triglyceride level (P<0.01), and triglyceride level and total cholesterol level decreased significantly in the middle dosage group (P<0.05-0.001). The results are shown in Table 16. -23- Table 16. Effects of the pharmaceutical composition of the present invention on blood fat of rats having diabetes mellitus type i (T±s) Group Dosage n Total cholesterol (mmol/I) Triglyceride (mmol/I) (g/kg) Control group - 12 1.47±0.25 1.05±0.32 Model group - 11 2.28±0.27 1.83±0.42 Metformin group 0.2 12 2.34±0.99 1.82±0.63 Jin Qi Jaing Tang tablet 2 8 2.16±0.36 1.52±0.59 group Large dosage group 8 11 2.05±0.56 1.18±0.49## Middle dosage group 4 10 2.01±0.31 # 0.92±0.58### Small dosage group 2 11 2.39±1.08 1.48±0.65 As compared to the control group: P<0.001; As compared to the model group: # P<0.05, P<0.01, P<0.001. 3.3. Effects of the pharmaceutical composition of the present invention on the indexes such as insulin level of rats having diabetes mellitus type 11 After the rats were subjected to the oral gavage of drugs for consecutive two months, as compared to the model group, their insulin level increased (P<0.05). As compared to the model group, the various dosage groups for the pharmaceutical composition of the present invention did not exhibit significant difference in the indexes such as tumor necrosis factor, interleukin-6, etc. The results are shown in Table 17. -24- Table 17. Effects of the pharmaceutical composition of the present invention on the indexes such as insulin level of rats having diabetes mellitus type II (±s) Group Dosage Tumor necrosis Interleukin -6 (g/kg) factor (ng/ml) (pg/ml) Control group 12 10.34±1.69 4.10±1.37 149.39±19.23 Model group - 11 9.24±2.39 5.04±1 55 191.07±36.64 ** Metformin group 0.2 12 13.29±3.53 # # 4.68±1.19 178.01±36.67 Jin Mao Jiang Tang 2 8 11.64±3.54 4.64±1.24 179.61±29.80 tablet group Large dosage group 8 11 11.41±1.64 # 4.32±1.90 193.52±79.02 Middle dosage group 4 10 10.37±1.25 4.16±0.75 234.76±64.22 Small dosage group 2 11 10.57±1.76 4.13±1.08 234.84±28.93## As compared to the control group: ** P<0.01; As compared to the model group: # P<0.05, P<0.01. 3.4. Effects of the pharmaceutical composition of the present invention on the body weight of rats having diabetes mellitus type II As compared to the control group, the body weight of all groups of rats decreased in different extent after the injection of STZ, and decreased significantly from the 2 nd week (P<0.05 -0.001). After the oral gavage for consecutive two months, the body weight of rats still did not reach the level before the models were established. The results are shown in Table 18 and 19. Table 18. Effects of the pharmaceutical composition of the present invention on the body weight of rats having diabetes mellitus type II (g, T±s) Before 1st week after 2nd week after 3rd week after 4th week after Group Dose n administration administration administration administration administration Control group - 12 306.33±61 00 315.17±63.92 319.17±68.46 315.58±62.61 327 75±69.55 Model group - 11 282.18±46.51 289 82±46 66 258 55±41.44* 269.64±45.55 253.91±44.61* Metformin group 0.2 12 284.42±56 72 290.33±51.60 263.75±46.22* 275.92±47.27 271.33±47.77* Jin Qi Jiang Tang tablet 2 8 281.25±42.93 295 67±44 08 262 33±32 69* 266.00±33 23* 261 08±31 70" group Large dosage group 8 11 278 83±49 15 291.00±46 15 257 18±40 42* 252 7±33 26" 256.73±37,60'' Middle dosage group 4 10 282 42±61 16 289 08±56 83 251 3±42 20" 264 42±44 81* 258 33±42 60" Small dosage group 2 11 279.42±48 83 276.17±33 55 237±29.98*" 247 4±31 12" 242 17±30 36'" -25- Table 19. Effects of the pharmaceutical composition of the present invention on the body weight of rats having diabetes mellitus type II (g, T±s) 5th week after 6th week after 7th week after 8th week after Group Dose n administration administration administration administration Control group - 12 329.42±70.84 333.58±72.38 339.67±74 63 340.83±74 60 Model group - 11 269.18±47.28' 243.1±65.37" 244.4±54.25" 234.64±56 95*" Metformin group 02 12 273 08±41 52* 261.0±36.23" 265.7±42.31" 230.17±38 87'" Jin Qi Jiang Tang tablet 2 8 262 91±37.20' 231.73±32.92* 242.27±31.47"* 242.25±22 98" group Large dosage group 8 11 261.7±34 33" 249.5±39.01 259.6±36.35" 230.09±2693"* Middle dosage group 4 10 265.58±51.72* 236 75±48.56" 244.9±51.73" 228.70±52 70*" Small dosage group 2 11 255.33±36.48" 244.82±39.38" 241.73±33.59"* 229 45±27.99"* As compared to the control group: * P<0.05, P<0.01, P<0.001 3.5. Effects of the pharmaceutical composition of the present invention on the food consumption of rats having diabetes mellitus type II As compared to the control group, the food consumption of all rats increased significantly after the injection of STZ, and exhibited significant difference from the 0 th week (P<0.05 -0.001, after the injection of STZ and before the administration of drugs). After the oral gavage for 3 weeks, as compared to the model group, the food consumption of rats of the various dosage groups for the pharmaceutical composition of the present invention decreased in different extents, and the difference was significant in the 4 th week (P<0.05), which indicated the decrease of blood sugar level and the alleviation of symptoms of diabetes mellitus. The results are shown in Table 20 and 21. -26- Table 20. Effects of the pharmaceutical composition of the present invention on the food consumption of rats having diabetes mellitus type II (g diet/100g body weight, T±s) Group n 0th week 1st week 2nd week 3rd week 4th week Control group 12 5.79±0.78 6.64±1.01 5.00±1.23 6.27±0.28 5.34±1.24 Model group 11 13.34±4.78 12 44±1.29* 18.86±0.88" 19 56±2 08' 21.93±0.62" Metformin group 12 14.26±2.82 11.46±3.90 15.17±2.13 14.73±0.20 13.67±0.63## Jin Qi Jiang Tang tablet 8 11.61±1.65* 12 78±0 72 13.79±0.88# 14 49±0 58 15.9±0.22## group Large dosage group 11 14.15±0 07** 14.39±0 86 18.92±2 75 17.10±1 49 18.48±0.08# Middle dosage group 10 13.28±0.67** 12 75±2.11 17 81±1 29 17 78±0.36 1749±2.08 Small dosage group 11 11.41±164* 12.68±0.70 16 92±1.14 16.03±1.28 15.13±1.22# Table 21. Effects of the pharmaceutical composition of the present invention on the food consumption of rats having diabetes mellitus type II (g diet/100g body weight, T±s) Group n 5th week 6th week 7th week 8th week Control group 12 3.59±0 35 5.31±1.34 4.40±0.07 4.78±0.11 Model group 11 18.30±0.94" 19.68±0.61" 21.56±3.21 20.48±4.22* Metformin group 12 13 38±1 79 15.31±1.59 1696±2.60 15.59±2.65 Jin Qi Jiang Tang tablet 8 14 12±0 51# 18.26±1.19 16.63±1 03 20.36±3.87 group Large dosage group 11 16 42±3.41 21.29±1.45 18.17±0.44 18.24±1.78 Middle dosage group 10 16 46±0 80 17.99±7.81 18.25±0.67 18.00±3.08 Small dosage group 11 16.83±1.76 17.54±1.65 16.78±1.30 16.71±1.89 As compared to the control group: * P<0.05, ** P<0.01; As compared to the model group: # P<0.05, ## P<0.01. 3.6. Effects of the pharmaceutical composition of the present invention on the organ index of diabetic rats The increase of organ indexes after the injection of STZ related to the loss of body weight. The results are shown in Table 22. -27- Table 22. Effects of the pharmaceutical composition of the present invention on the organ index of rats having diabetes mellitus type II (unit: g/1OOg body weight, x±s) Group Dosage n Heart index Liver index Kidney index Control group - 12 0.344±0.068 2.395±0.122 0.292±0.038 Model group - 11 0.416±0.043** 4.549±0.393*** 0.488±0.049*** Metformin group 0.2 12 0.438±0.041*** 4.900±0.394*** 0.471±0.053*** Jin Qi Jiang Tang tablet group 2 8 0.402±0.029* 4.856±0.253*** 0.499±0.058*** Large dosage group 8 11 0.443±0.067** 4.954±0.603*** 0.483±0.050*** Middle dosage group 4 12 0.396±0.039* 4.731±0.508*** 0.466±0.026*** Small dosage group 2 11 0.437±0.051** 4.810±0.304*** 0.483±0.043*** As compared to the control group: * P<0.05, ** P<0.01, *** P<0.001. 4. Conclusion The above results show that the insulin resistance was successfully induced in rats by feeding rats with a high-fat and high-sugar diet up to one month when hyperinsulinism accompanied by increased blood cholesterol and sugur. The diabetes mellitus type 11 rat models were established by intraperitoneal injection of STZ. After oral gavage of drugs for consecutive two months, blood sugar level of rats in the large dosage group decreased significantly, and the rate of blood sugar decrease was 31.23%. The blood fat level of rats in the large dosage group and the middle dosage group decreased significantly. Thus, the pharmaceutical composition of the present invention exhibits effects of reducing both blood sugar and fat in rats having diabetes mellitus type 11. Example 6: Experimental study of the pharmaceutical composition of the present invention in rats with quickly onset diabetes mellitus (diabetes mellitus type 1) 1. Experimental material 1.1. Animals Wistar grade 11 male rats with a body weight of 180-200g were provided by the Institute of Laboratory Animals, Chinese Academy of Medical Sciences, License No. SCXK (Beijing) 2000-0006. The rats were bred in the Center of Laboratory Animals -28of Xiyuan Hospital of Chinese Academy of Medical Sciences, Second grade animal breading facility, Certificate No. SYXK (Beijing) 2003-0008. Room temperature: 22-25*C, relative humidity: 45-60%. 1.2. Diets Common diets were provided by Beijing Ke Ao Xie Li Diets Company, product license No. 238, standard: GB14924, 3-2001. Essential nutrients: crude protein >18%, crude fibers 55%, crude ash 58%, calcium 0.8-1.8%, phosphorus 0.6-1.2%, lysine 21.35%, sodium chloride 0.5%, water 510%, various vitamins and trace elements. 1.3. Drugs The pharmaceutical composition of the present invention: extract paste, 3.37g crude herb per gram of paste, batch No. 050401, prepared in accordance with the method of Example 4. Metformin hydrochloride tablets: 0.25g/tablet, batch No. 041226, Beijing Li Ling Heng Tai Pharmaceutical Company, H11021560. Jin Qi Jiang Tang tablets: 0.42g/tablet, batch No. 0503746, produced by Long Shun Rong Pharmaceutical Factory of Tianjin Zhong Xin Pharmaceutical Group Company, Z10920027. 1.4. Reagents Citric acid and sodium citrate: Beijing Chemical Plant, batch No. 960904. Streptozotocin (STZ): provided by Sigma Company, batch No. S0130 and subpackaged by Beijing Tian Lai Biomedicine Science Company. Blood sugar test paper: produced by Roche Company, batch No.22898731, and subpackaged by Roche-Diagnostics (Shanghai) Company, Ltd. Insulin radio-immunity kits: Beijing Fu Rui Bioengineering Company, batch 200601. Glucagon radio-immunity kits: Beijing Atomic High-Tech Nuclear Technology Application Corporation, batch No.200601. Glycosylated hemoglobin: produced by Roche Company, batch No.200512. 2. Experimental methods Wistar grade 11 male rats with a body weight of 180-200g were used. Except rats of the blank control group, all rats were injected with streptozotocin (STZ) via caudal vein (90mg/kg, prepared by using 0.1M citric acid buffer) to establish -29models. The rats with a fasting blood sugar of 16.7mmol/l measured after 72 h were deemed as successful models, and were divided into the following groups: (1) blank control group; (2) model group; (3) positive metformin group (0.2g/kg); (4) positive Jin Mao Jiang Tang tablet group (2g/kg); (5) the pharmaceutical composition of the present invention, large dosage group (8g crude herb/kg); (6) the pharmaceutical composition of the present invention, middle dosage group (4g crude herb/kg); (7) the pharmaceutical composition of the present invention, small dosage group (2g crude herb/kg). Except the control group and the model group, the rats of other groups were subjected to oral gavage of drugs in determined dosages. Body weight was measured every week, and blood sugar was measured every other week. Whole blood samples (25ul EDTA + 1ml whole blood anticoagulation) were obtained from the abdominal aorta of the anesthetized rats to determine glycosylated hemoglobin, blood serum was separated to determine insulin, and plasma (25ul EDTA + 1 ml whole blood anticoagulation) was separated to determine glucagons. Pancreas was used to make pathological slices (HE stain, and aldehyde-fuchsin specific stain). The results were subjected to statistical treatment (t-test). 3. Results 3.1. Effects of the pharmaceutical composition of the present invention on blood sugar of rats having quickly onset diabetes mellitus The rats were injected with STZ via caudal vein, then their fasting blood sugar levels were measured after 72 h, and as compared to the control group, the blood sugar increased significantly (P<0.001) in the groups before the administration of drugs, and the average blood sugar level was about 23-24mmol/l, which means the models were successfully established. The rats of the groups were subjected to oral gavage of drugs for two consecutive months. As compared to the model group, the blood sugar of rats of the large dosage group for the pharmaceutical composition of the present invention significantly decreased in the 6 'h to 8 th weeks (P<0.05), and the small dosage group showed hypoglycemic effects in the 8 th week (P<0.05). The results are shown in Table 23. -30- Table 23. Effects of the pharmaceutical composition of the present invention on blood sugar of rats having quickly onset diabetes mellitus (iv±s) Blood sugar (mmol/) Group n Before 2 week after 4 week after 6 week after 8 week after administration administration administration administration administration Control group 13 6.53±0.43 6 32±0 32 6.46±0.53 6.39±0.43 5.39±0.32 Model group 13 23.27±3.48*" 29.38±3.48'" 29.85±2.17*" 28.75±1.40"' 28.99±2.30"' Metformin group 12 23.61±2.38'" 25.91±2.61 ## 25.36±4.83 ## 18.43±9.36 ### 22.51±3.50 ### Jin Qi Jiang Tang table group 11 24.08±2.69"* 27 23±2.50 26.82±5.45 27.42±3.88 26.66±3.53 Large dosage group 11 23.132.64"* 28.30±5.24 27.25±5.17 24.98±5.37 # 25.02±5.72 # Middle dosage group 12 23.50±2.92*" 29.50±3 25 29.11±2.62 26.88±2.02 # 25.58±4.50 # Small dosage group 10 23.64±3.44'" 29.20±3.21 28.58±1.73 28.34±2.09 25.50±5.36 # As compared to the control group: *** P<0.001; As compared to the model group: # P<0.05, ## P<0.01, ### P<0.001. 3.2. Effects of the pharmaceutical composition of the present invention on glycosylated hemoglobin of rats having quickly onset diabetes mellitus After oral gavage of drugs for consecutive two months, as compared to the model group, the large dosage group for the pharmaceutical composition of the present invention exhibited a decrease of glycosylated hemoglobin (HBAIc) (P<0.05) and a significant decrease of percentage of glycosylated hemoglobin (Halc%) (P<0.01), but the hemoglobin (HB) of the group was not significantly different from that of the model group. The results are shown in Table 24. Table 24. Effects of the pharmaceutical composition of the present invention on glycosylated hemoglobin of rats having quickly onset diabetes mellitus (i±s) Group Dosage n Halc % HBAlc HB Control group - 13 3.60±0.08 0.27±0.04 17.94±3.14 Model group - 13 7.83±0.47*** 1.10±0.19*** 17.35±2.51 Metformin group 0.2 g/kg 12 7.38±0.47# 0.92±0.16# 15.69±2.00 Jin Qi Jiang Tang tablet group 2 g/kg 11 7.39±0.73 1.02±0.17 17.36±1.35 Large dosage group 8 g/kg 11 7.04±0.80## 0.92±0.20# 16.81±1.36 Middle dosage group 4 g/kg 12 7.43±0.73 1.04±0.21 17.60±1.80 Small dosage group 2 g/kg 10 7.59±0.79 1.06±0.25 17.36±3.24 -31- As compared to the control group: * P<0.05, *** P<0.001; As compared to the model group: # P<0.05, ## P<0.01. 3.3. Effects of the pharmaceutical composition of the present invention on insulin and glucagons of rats having quickly onset diabetes mellitus After oral gavage of drugs for consecutive two months, as compared to the model group, insulin level had a trend of increase but had no statistical significance. As compared to the model group, the large dosage group exhibited a decrease in glucagons (P<0.05). The results are shown in Table 25. Table 25. Effects of the pharmaceutical composition of the present invention on insulin and glucagons of rats having quickly onset diabetes mellitus (x±s) Group Dosage n Insulin (ulU/ml) Glucagons (pg/ml) (g/kg) Control group - 13 14.92±7.18 219.72±42.88 Model group - 13 4.72±1.79 *** 445.63±134.52 Metformin group 0.2 12 5.51±2.21 337.35±70.83 # Jin Qi Jiang Tang tablet group 2 11 4.38±1.81 349.39±96.65 Large dosage group 8 11 4.88±1.23 350.35±73.99 # Middle dosage group 4 12 5.34±1.49 380.11±130.68 Small dosage group 2 10 4.85±0.85 406.54±140.05 As compared to the control group: *** P<0.001; As compared to the model group: # P<0.05. 3.4. Effects of the pharmaceutical composition of the present invention on body weight of rats having quickly onset diabetes mellitus As compared to the control group, the loss of body weight was observed in each group of rats after the injection of STZ, and the body weight decreased significantly from the 2 nd week (P<0.05-0.001). After oral gavage of drugs for consecutive two months, the body weight of rats was still less than that before the models were established. The results are shown in Table 26. -32- Table 26. Effects of the pharmaceutical composition of the present invention on body weight of rats having quickly onset diabetes mellitus (g, -±s) Group 2 week after 4 week after 6 week after 8 week after Before administration administration administration administration administration Controlgroup 227.00=8.75 312.46=16.01 366.39± 23.19 387.92 26.74 421.31 ±35.39 Model group 202.23=11.31 227.31 19.64 254.62=27.96 233.31 32. 11 240.15 ±35.56 Metformingroup 198.62 14.84 217.77 23.46 222.00±23.03 206.58*33.08 204.17i41.23 Jin Qi JiangTang tablet group 199.46 9.58 216.83±23.47 206.00*21.47 201.82 24.09 216.85 12 69 Largedosagegroup 195.21 17.90 213.86±31.37 212.57 25.40 220.23=39.05 213.82*37.08 Middledosagegroup 196.69 10.65 205.69+21.28 224.69± 22.01 207. 25=32. 76 208. 25±37.71 Small dosage group 197. 69=15.85 200.00 34 05 201 .20 41 .36 210.50 ±37. 024 194.50±41.90## As compared to the control group: *** P<0.001; as compared to the model group: #P<0.05, ## P<0.01. 3.5. Effects of the pharmaceutical composition of the present invention on food consumption of rats having quickly onset diabetes mellitus As compared to the control group, the food consumption of each group of rats increased significantly after the injection of STZ, the difference was significant from the 0 th week (after the injection of STZ, but before the administration of drugs) (P<0.05 -0.001), and the diabetic symptoms of polyphagia and polydipsia were obvious. As compared to the model group, the large and middle dosage groups for the pharmaceutical composition of the present invention decreased in various extents after weeks of oral gavage of drugs, but the difference was not statistically significant. With the decrease of blood sugar level, the diabetic symptoms were alleviated. The results are shown in Table 27. -33- Table 27. Effects of the pharmaceutical composition of the present invention on food consumption of rats having quickly onset diabetes mellitus (g, T±s) Group 0 week after 2 week after 4 week after 6 week after 8 week after dosing dosing dosing dosing dosing Control group 11.57±0.36 9.44±0.41 8.22±0.02 7.55±0.57 6 63±0.43 Model group 37.66±3.22"* 35.62±7.08" 28.97±6.02" 26.40±8.22* 28.37±4.14" Metformin group 36.19±1.74*" 26.21±2 46" 24.76±3.21" 23.63±2.64" 23.89±1.22*" Jin Qi Jiang Tang tablet group 33.81±3.67" 23.90±6.13* 21.82±744 20.85±1.76"* 29 78±4.19" Large dosage group 34.07±5.10" 25.03±1.27*" 24.22±2.77" 19.11±3.73* 25.14±1.74"* Middle dosage group 37.82±4.27*" 31.49±4.83" 29.87±4.79" 20.31±6.81 29.25±1 26*" Small dosage group 38.68±5.47" 36.13±7.77" 32 95±1.78*" 31.66±2.07*" 32 61±4 58" As compared to the control group: * P<0.05, ** P<0.01, P<0.001. 4. Conclusion A model for quickly onset diabetes mellitus (diabetes mellitus type I) was established in rats after they were injected with STZ. After oral gavage for consecutive two months, the blood sugar levels of rats in all dosage groups of the pharmaceutical composition of the present invention decreased significantly. As compared with the model group, the glycosylated hemoglobin and the percentage of glycosylated hemoglobin decreased significantly in the large dosage group, and the glucagon also decreased somewhat in the large dosage group. Although insulin level exhibited a trend of increase and food consumption decreased in some extent, they are not statistically significant. Thus, the pharmaceutical composition of the present invention exhibits effects of reducing blood sugar level in rats having quickly onset diabetes mellitus. -34-

Claims (9)

1. A pharmaceutical composition for regulating blood sugar and fat, comprising the following Chinese herbs or extracts thereof in weight parts: Ligustrum lucidum (Fructus Ligustri Lucidui) 5-17, Mongolian mikvetch root (Radix Astragali Mongolici)

3-12, Chinese goldthread rhizome (Rhizoma Coptidis) 1-5 and Lychee seed (Semen Litchi) 1-5, and pharmaceutically acceptable excipients. 2. A pharmaceutical composition for regulating blood sugar and fat, consisting of the following Chinese herbs or extracts thereof in weight parts: Ligustrum lucidum (Fructus Ligustri Lucidui) 5-17, Mongolian mikvetch root (Radix Astragali Mongolici) 3-12, Chinese goldthread rhizome (Rhizoma Coptidis) 1-5, Lychee seed (Semen Litchi) 1-5, Kelp (Tha/us Laminariae Japonicae) 1-6, and Common turmeric rhizome (Rhizoma Curcumae Longae) 1-6. 3. The pharmaceutical composition according to claim 2, consisting of the following Chinese herbs or extracts thereof in weight parts: Ligustrum lucidum 8-15, Mongolian mikvetch root 4-10, Chinese goldthread rhizome 2-3, Lychee seed 3-4, Kelp 3-5, and Common turmeric rhizome 3-5.

4. The pharmaceutical composition according to claim 2, consisting of the following Chinese herbs or extracts thereof in weight parts: Ligustrum lucidum 8, Mongolian mikvetch root 4, Chinese goldthread rhizome 2, Lychee seed 4, Kelp 3, and Common turmeric rhizome 3.

5. The pharmaceutical composition according to any one of claims 1 to 4, wherein the extracts are alcohol extracts, water extracts or volatile oils.

6. The pharmaceutical composition according to any one of claims 1 to 4, characterized in that, when detected using the thin layer chromatography in the Annex VIB of the Pharmacopoeia of the People's Republic of China, Volume one, 2005 Edition, is adopted and oleanolic acid, berberine hydrochloride, Astragaloside -35- and protocatechuic acid are respectively used as controls, the pharmaceutical composition presents spots with same colors as those of the controls at the corresponding positions.

7. The pharmaceutical composition according to any one of claims 1 to 4, characterized in that, when detected using the thin layer chromatography in the Annex VIB of the Pharmacopoeia of the People's Republic of China Volume one, 2005 Edition, is adopted and oleanolic acid and ursolic acid are used as controls, the contents of oleanolic acid and ursolic acid in the pharmaceutical composition are not less than 4.0mg and 1.0mg, respectively.

8. A method for preparing a pharmaceutical composition according to any one of claims 1 to 4, comprising the following steps: Extracting Ligustrum lucidum with ethanol by heating under reflux for several times, combining ethanol extracts, recovering ethanol, and drying the extract for standby; Extracting Common turmeric rhizome by water steam distillation, collecting a volatile oil, a water solution and a residue for standby; Including the obtained volatile oil with cyclodextrin and water, stirring, freezing, filtering, cryodrying the obtained inclusion, and pulverizing for standby; Decocting lychee seed and kelp with water for several times, combining decocting solutions, filtering, combining the filtrate and the water solution of Common turmeric rhizome, concentrating, adding alcohol, freezing, filtering to obtain a water decocting-alcohol settling solution for standby; and Extracting Chinese goldthread rhizome, Mongolian milkvetch root and the residue of Common turmeric rhizome with alcohol by heating under reflux for several times, combining alcohol extracts, combining it with the water decocting-alcohol settling solution, recovering alcohol, drying, mixing homogeneously with the alcohol extract of Ligustrum lucidum and the fine powder of the cyclodextrin inclusion of Common turmeric rhizome, mixing optionally with pharmaceutically acceptable excipients to form a desired dosage form. -36-

9. A pharmaceutical composition for regulating blood sugar and fat, which is obtained in accordance with the method of claim 8.

10. Use of the pharmaceutical composition according to any one of claims 1 to 4 in the manufacture of a medicament for the treatment of diabetes mellitus and complications thereof. -37-