Combination product containing limonoid compound and biguanide compound

Abstract

The present invention relates to a combination product comprising a limonoid compound (or a pharmaceutically acceptable derivative, ester, stereoisomer, salt or prodrug thereof), and a biguanide compound (e.g., metformin, metformin hydrochloride, buformin, and phenformin). The present invention further relates to a use of the combination product for prevention and/or treatment of a disease associated with diabetes, for lipid-lowering and weight-loss, and the like.

Claims

1. A combination product, the combination product comprising a limonoid compound, or a pharmaceutically acceptable derivative or salt thereof, and a biguanide compound, or a pharmaceutically acceptable salt thereof, wherein the limonoid compound, or pharmaceutically acceptable derivative or salt thereof, is limonin, isolimonic acid, limonin 17-β-D-glucopyranoside, isolimonic acid 17-β-D-glucopyranoside, obacunone, obacunone 17-β-D-glucopynoside, ichangin, ichangensin, ichangin 17-β-D-glucopyranoside, nomilin, nomilin acid, deacetylnomilin, deacetylnomilin glucopyranoside, isoobacunoic acid, nomilin 17-β-D-glucopyranoside, or nomilinic acid 17-β-D-glucopyranoside, and the biguanide compound, or pharmaceutically acceptable salt thereof, is metformin or metformin hydrochloride.

2. The combination product according to claim 1, wherein the combination product is in the form of a pharmaceutical composition.

3. The combination product according to claim 1, wherein the limonoid compound, or a pharmaceutically acceptable derivative or salt thereof, and the biguanide compound, or a pharmaceutically acceptable salt thereof, are each in the form of a separate preparation.

4. The combination product according to claim 3, wherein the limonoid compound or a pharmaceutically acceptable derivative or salt thereof, and the biguanide compound, or a pharmaceutically acceptable salt thereof, are configured to be administered simultaneously or sequentially.

5. The combination product according to claim 1, wherein the biguanide compound, or a pharmaceutically acceptable salt thereof, is present in the combination product in an amount of 50 mg to 2000 mg.

6. The combination product according to claim 1, wherein the limonoid compound, or a pharmaceutically acceptable derivative or salt thereof, is present in the combination product in an amount of 50 mg to 2000 mg.

7. The combination product according to claim 1, wherein the combination product further comprises a pharmaceutically acceptable carrier, diluent, or excipient.

8. The combination product according to claim 7, wherein the combination product is in the form of a tablet, capsule, granule, syrup, powder, lozenge, sachet, cachet, elixir, suspension, emulsion, solution, syrup, aerosol, ointment, cream, or injection.

Description

EXAMPLE 1

(1) Effects of a Limonoid Compound, Metformin or a Combination Thereof on Blood Glucose in a Mouse Pancreatic Islet β-Cell Injury Model

(2) In this example, a mouse pancreatic islet β-cell injury model was established by modeling ICR mice with streptozotocin (STZ) (referred to the prior art literature: Li Nan et al., Protective effect of pine pollen on kidney damage in diabetic nephropathy mice, Science and Technology Review, 2014, 32 (4/5): 95-99), and used to complete the evaluation of hypoglycemic effect in animals (this model could simulate pancreatic islet β-cell damage state of type I and type II diabetics). The limonoid compound was selected from the group consisting of limonin, isolimonic acid, limonin 17-β-D-glucopyranoside, and isolimonic acid 17-β-D-glucopyranoside, and a metformin single administration group, limonin single administration group, isolimonic acid single administration group, limonin 17-β-D-glucopyranoside singe administration group, isolimonic acid 17-β-D-glucopyranoside singe administration group, and combination thereof with metformin administration groups were set, respectively.

(3) Conditions of experimental feeding: ICR mice (20±2 g), aged 6 weeks, purchased from Zhejiang Academy of Medical Sciences, and subjected to experimental feeding after 7 days of preliminary feeding. It should be noted that the conditions for raising the mice were as follows: the temperature was 23±1° C., the humidity was 55±10%, the lights were turned on between 7 am and 7 pm (the lights were turned off at other time), and the mice were allowed to access to water and feed freely. The experimental feed was mouse growth-stable feed (GB M2118), and the daily feeding and management of the animals were under the responsibility of the animal security department, which provided the animals with sufficient padding and fresh drinking water daily.

(4) Experimental grouping: 15 male mice were randomly selected as the normal control group. After fasting for 12 hours, the remaining mice were intraperitoneally injected once with STZ at a dose of 150 mg/kg, and 72 hours later, the mice with blood glucose value of 15 to 25 mmol/L were undifferentiatedly grouped and used in the experiment, 15 animals in each group, and subjected to blood sampling and detection of indicators after two weeks of administration.

(5) Gavage doses: the gavage dose was 0.02 g/kg per day for the limonin group, the gavage dose was 0.02 g/kg per day for the isolimonic acid group, the gavage dose was 0.02 g/kg per day for the limonin 17-β-D-glucopyranoside group, the gavage dose of metformin was 0.02 g/kg per day for the metformin group, limonin at a dose of 0.01 g/kg and metformin at a dose of 0.01 g/kg were simultaneously gavaged per day for the limonin/metformin combination group, isolimonic acid at a dose of 0.01 g/kg and metformin at a dose of 0.01 g/kg were simultaneously gavaged per day for the isolimonic acid/metformin combination group, limonin 17-β-D-glucopyranoside at a dose of 0.01 g/kg and metformin at a dose of 0.01 g/kg were simultaneously gavaged per day for the limonin 17-β-D-glucopyranoside/metformin combination group, isolimonic acid 17-β-D-glucopyranoside at a dose of 0.01 g/kg and metformin at a dose of 0.01 g/kg were simultaneously gavaged per day for the isolimonic acid 17-β-D-glucopyranoside/metformin combination group, the gavage volume was 10 mL/kg, and the normal group and the model group were administrated with 10 mL/kg of distilled water. Two weeks later, the blood glucose values were measured by tail trimming method (Johnson's blood glucose meter) 1 h after the last administration, and the average of each group was obtained. SPSS 16.0 software was used for statistical analysis. The data were expressed as mean and standard deviation. The data before and after were analyzed by t-test, and P<0.05 was considered statistically significant. The test results were shown in Table 1 below.

(6) TABLE-US-00001 TABLE 1 Blood glucose values of STZ mice after two weeks of daily intragastric gavage. Blood glucose Formulation and dose value Group of administration (mmol/L) Normal control None 7.5 ± 0.61 group Model group None 29.5 ± 4.6  Metformin group Metformin 0.02 g/kg 22.6 ± 3.9** Limonin group Limonin 0.02 g/kg 19.5 ± 1.8** Isolimonic acid Isolimonic acid 0.02 18.2 ± 3.1** group g/kg Limonin 17-β-D- Limonin 17-β-D- 18.9 ± 2.2** glucopyranoside glucopyranoside 0.02 group g/kg Isolimonic acid Isolimonic acid 17-β-D- 17.9 ± 2.8** 17-β-D- glucopyranoside 0.02 g/kg glucopyranoside group Limonin/metformin Limonin 0.01 g/kg +  8.8 ± 3.1** combination group metformin 0.01 g/kg Isolimonic acid/ Isolimonic acid 0.01 g/kg +  9.2 ± 3.3** metformin combi- metformin 0.01 g/kg nation group Limonin 17-β-D- Limonin 17-β-D-  9.1 ± 3.6** glucopyranoside/ glucopyranoside 0.01 g/kg + metformin combi- metformin 0.01 g/kg nation group Isolimonic acid Isolimonic acid 17-β-D-  9.4 ± 2.3** 17-β-D- glucopyranoside 0.01 g/kg + glucopyranoside/ metformin 0.01 g/kg metformin combi- nation group Note * After independent t-test, compared with the model group, the difference was extremely significant (P < 0.05) **After independent t-test, compared with the model group, the difference was extremely significant (P < 0.01)

(7) Discussion of Experimental Results

(8) From the above results, it could be seen that, compared with the model group, either in single administration or in combination administration with metformin, limonin and its derivatives could significantly reduce the blood glucose values of the mice with STZ pancreatic islet cell injury. The administration of limonin and its derivatives in combination with metformin had significantly improved the effect as compared with their single administration, showing a synergistic effect. In addition, when limonin and its derivatives were administrated in combination with metformin, as compared with their single administration, the doses of both could be effectively reduced while comparable glucose-lowering effects could still be achieved, which improved the safety of therapeutic regimen and reduced side effects.

EXAMPLE 2

(9) Effects of a Limonoid Compound, Metformin or a Combination Thereof on Blood Glucose and Leptin in a Mouse Model of Type II Diabetes

(10) In the present example, db/db mice (line name BKS.Cg-Dock7.sup.m+/+ Lepr.sup.db/Nju) were used to perform hypoglycemic efficacy evaluation test of animals (blood glucose level and leptin). The limonoid compound is selected from obacunone, isoobacunoic acid and obacunone 17-β-D-glucopynoside, and an obacunone single administration group, isoobacunoic acid single administration group, obacunone 17-β-D-glucopynoside single administered group, and combination thereof with metformin administration groups were set, respectively.

(11) Conditions for experimental feeding: as type II diabetes model mice, 6-week-old SPF-grade db/db mice were purchased from the Nanjing Model Biology Institute, and subjected to experimental feeding after 7 days of preliminary feeding. It should be noted that the conditions for raising the mice were as follows: the temperature was 23±1° C., the humidity was 55±10%, the lights were turned on between 7 am and 7 pm (the lights were turned off at other time), and the mice were allowed to access to water and feed freely. The experimental feed was mouse growth-stable feed (GB M2118), and the daily feeding and management of the animals were under the responsibility of the animal security department, which provided the animals with sufficient padding and fresh drinking water daily.

(12) Experimental grouping: male db/db mice (20±2 g) were selected, and 18 male mice in each group were tested. The experimental groups included the normal control group (db/m, n=18), the model group (db/db, n=18), the obacunone group (db/db, n=18), the isoobacunoic acid group (db/db, n=18), the obacunone 17-β-D-glucopynoside group (db/db, n=18), the metformin group (db/db, n=18), the obacunone/metformin combination group (db/db, n=18), the isoobacunoic acid/metformin combination group (db/db, n=18), the obacunone 17-β-D-glucopynoside/metformin combination group (db/db, n=18).

(13) Gavage doses: obacunone at a dose of 0.04 g/kg was gavaged per day for the obacunone group, isoobacunoic acid at a dose of 0.04 g/kg was gavaged per day for the isoobacunoic acid group, obacunone 17-β-D-glucopynoside at a dose of 0.04 g/kg was gavaged per day for the obacunone 17-β-D-glucopynoside group, metformin at a dose of 0.04 g/kg was gavaged per day for the metformin group, obacunone at a dose of 0.02 g/kg and metformin at a dose of 0.02 g/kg were simultaneously gavaged per day for the obacunone/metformin combination group, isoobacunoic acid at a dose of 0.02 g/kg and metformin at a dose of 0.02 g/kg were simultaneously gavaged per day for the isoobacunoic acid/metformin combination group, obacunone 17-β-D-glucopynoside at a dose of 0.02 g/kg and metformin at a dose of 0.02 g/kg were simultaneously gavaged per day for the obacunone 17-β-D-glucopynoside/metformin combination group, the gavage volume was 10 mL/kg, and the normal group and the model group were administrated with 10 mL/kg of distilled water. Two weeks later, the blood glucose values were measured by tail trimming method (Johnson's blood glucose meter) 1 h after the last administration, and serum leptin levels were measured with blood collected from orbital cavity by enzyme-linked immunosorbent assay (Elisa), and the average of each group was obtained. SPSS 16.0 software was used for statistical analysis. The data were expressed as mean and standard deviation. The data before and after were analyzed by t-test, and P<0.05 was considered statistically significant. The test results were shown in Table 2 below.

(14) TABLE-US-00002 TABLE 2 Blood glucose values and leptin of db/db mice after two weeks of daily gavage. Formulation and dose Blood glucose Leptin Group of administration value (mmol/L) (pg/ml) Normal control group None  6.0 ± 0.68 0.88 ± 0.18  Model group None 23.9 ± 4.2  48.19 ± 8.12  Obacunone group Obacunone 0.04 g/kg 12.71 ± 2.8** 25.99 ± 3.92** Isoobacunoic acid group Isoobacunoic acid 0.04 g/kg 14.66 ± 3.1** 27.68 ± 3.25** Obacunone 17-β-D- Obacunone 17-β-D- 13.67 ± 2.9** 28.55 ± 4.00** glucopynoside group glucopynoside 0.04 g/kg Metformin group Metformin 0.04 g/kg  15.5 ± 3.6** 34.49 ± 3.38** Obacunone/metformin Obacunone 0.02 g/kg +  6.9 ± 2.2** 18.42 ± 4.92** combination group metformin 0.02 g/kg Isoobacunoic Isoobacunoic acid 0.02 g/kg +  7.5 ± 1.6** 19.89 ± 3.67** acid/metformin metformin 0.02 g/kg combination group Obacunone 17-β-D- Obacunone 17-β-D-  7 3 ± 2.1** 19.48 ± 3.71** glucopynoside/metformin glucopynoside 0.02 g/kg + combination metformin 0.02 g/kg Note **after independent t-test, compared with the model group, the difference was extremely significant (P < 0.01) * after independent t-test, compared with the model group, the difference was extremely significant (P < 0.05)

(15) Discussion of Experimental Results

(16) From the above results, it could be seen that, compared with the model group, either in single administration or in combination administration with metformin, obacunone and derivatives thereof could significantly reduce the blood glucose levels in the db/db diabetic mice. When obacunone and derivatives thereof were administrated in combination with metformin, significantly improved effect was observed relative to the single administration thereof, showing a synergistic effect. In addition, when obacunone and derivatives thereof were administrated in combination with metformin, as compared with their single administration, the doses of both could be effectively reduced while comparable glucose-lowering effects could still be achieved, which improved the safety of therapeutic regimen and reduced side effects.

(17) Meanwhile, the limonoid compound represented by obacunone and its derivatives could significantly improve the sensitivity to leptin; and especially when administrated in combination with metformin, it could significantly improve the utilization efficiency of leptin in the body, improve the glucose metabolism of the body, and improve the functions relevant to the glucose metabolism in diabetes mice.

EXAMPLE 3

(18) Effects of a Limonoid Compound, Metformin or a Combination Thereof on Blood Glucose in a Mouse Pancreatic Islet β-Cell Injury Model

(19) In this example, a mouse pancreatic islet β-cell injury model was established by modeling ICR mice with streptozotocin (STZ) (referred to the prior art literature: Li Nan et al., Protective effect of pine pollen on kidney damage in diabetic nephropathy mice, Science and Technology Review, 2014, 32 (4/5): 95-99), and used to complete the evaluation of hypoglycemic effect in animals (this model could simulate pancreatic islet β-cell damage state of type I and type II diabetics). The limonoid compound was selected from the group consisting of ichangin, ichangensin, and ichangin 17-β-D-glucopyranoside, and a metformin single administration group, ichangin single administration group, ichangensin single administration group, ichangin 17-β-D-glucopyranoside singe administration group, and combination thereof with metformin administration groups were set, respectively.

(20) Conditions of experimental feeding: ICR mice (20±2 g), aged 6 weeks, purchased from Zhejiang Academy of Medical Sciences, and subjected to experimental feeding after 7 days of preliminary feeding. It should be noted that the conditions for raising the mice were as follows: the temperature was 23±1° C., the humidity was 55±10%, the lights were turned on between 7 am and 7 pm (the lights were turned off at other time), and the mice were allowed to access to water and feed freely. The experimental feed was mouse growth-stable feed (GB M2118), and the daily feeding and management of the animals were under the responsibility of the animal security department, which provided the animals with sufficient padding and fresh drinking water daily.

(21) Experimental grouping: 15 male mice were randomly selected as the normal control group. After fasting for 12 hours, the remaining mice were intraperitoneally injected once with STZ at a dose of 150 mg/kg, and 72 hours later, the mice with blood glucose value of 15 to 25 mmol/L were undifferentiatedly grouped and used in the experiment, 15 animals in each group, and subjected to blood sampling and detection of indicators after two weeks of administration.

(22) Gavage doses: the gavage dose was 0.1 g/kg per day for the ichangin group, the gavage dose was 0.1 g/kg per day for the ichangensin group, the gavage dose was 0.1 g/kg per day for the ichangin 17-β-D-glucopyranoside group, the gavage dose of metformin was 0.1 g/kg per day for the metformin group, ichangin at a dose of 0.05 g/kg and metformin at a dose of 0.05 g/kg were simultaneously gavaged per day for the ichangin/metformin combination group, ichangensin at a dose of 0.05 g/kg and metformin at a dose of 0.05 g/kg were simultaneously gavaged per day for the ichangensin/metformin combination group, ichangin 17-β-D-glucopyranoside at a dose of 0.05 g/kg and metformin at a dose of 0.05 g/kg were simultaneously gavaged per day for the ichangin 17-β-D-glucopyranoside/metformin combination group, the gavage volume was 10 mL/kg, and the normal group and the model group were administrated with 10 mL/kg of distilled water. Two weeks later, the blood glucose values were measured by tail trimming method (Johnson's blood glucose meter) 1 h after the last administration, and the average of each group was obtained. SPSS 16.0 software was used for statistical analysis. The data were expressed as mean and standard deviation. The data before and after were analyzed by t-test, and P<0.05 was considered statistically significant. The test results were shown in Table 3 below.

(23) TABLE-US-00003 TABLE 3 Blood glucose values of STZ mice after two weeks of daily gavage. Blood glucose Formulation and dose value Group of administration (mmol/L) Normal control group None 7.5 ± 0.61 Model group None 29.5 ± 4.6  Metformin group Metformin 0.1 g/kg 17.2 ± 3.9** Ichangin group Ichangin 0.1 g/kg 14.8 ± 1.9** Ichangensin group Ichangensin 0.1 g/kg 14.5 ± 3.5** Ichangin 17-β-D- Ichangin 17-β-D- 14.9 ± 2.1** glucopyranoside glucopyranoside 0.1 g/kg group Ichangin/metformin Ichangin 0.05 g/kg +  7.7 ± 4.1** combination group metformin 0.05 g/kg Ichangensin/ Ichangensin 0.05 g/kg +  7.6 ± 2.3** metformin combi- metformin 0.05 g/kg nation group Ichangin 17-β-D- Ichangin 17-β-D-  8.0 ± 1.7** glucopyranoside/ glucopyranoside 0.05 g/kg + metformin combi- metformin 0.05 g/kg nation group Note **After independent t-test, compared with the model group, the difference was extremely significant (P < 0.01) * After independent t-test, compared with the model group, the difference was extremely significant (P < 0.05)

(24) Discussion of Experimental Results

(25) From the above results, it could be seen that, compared with the model group, either in single administration or in combination administration with metformin, the three limonoid compounds all could significantly lower the blood glucose levels in the mice of the STZ pancreatic islet cell injury model. When they were administrated in combination with metformin, their effects were significantly increased as compared with their single administration, similar to the normal mice in blood glucose level, showing a synergistic effect. In addition, when the above three limonoid compounds were administration in combination with metformin, as compared with their single administration, the doses of both could be effectively reduced while comparable glucose-lowering effects could still be achieved, which improved the safety of therapeutic regimen and reduced side effects.

EXAMPLE 4

(26) Effects of a Limonoid Compound, Metformin or a Combination Thereof on Blood Glucose and Insulin in a Mouse Model of Type II Diabetes

(27) In the present embodiment, the limonoid compound selected nomilin, deacetylnomilin, nomilin acid, deactylnomilin 17-β-D-glucopyranoside, and a nomilin single administration group, deacetylnomilin single administration group, nomilin acid single administered group, deactylnomilin 17-β-D-glucopyranoside single administration group, and combination thereof with metformin administration groups were set, respectively.

(28) Conditions for experimental feeding: as type II diabetes model mice, 6-week-old SPF-grade db/db mice were purchased from the Nanjing Model Biology Institute, and subjected to experimental feeding after 7 days of preliminary feeding. It should be noted that the conditions for raising the mice were as follows: the temperature was 23±1° C., the humidity was 55±10%, the lights were turned on between 7 am and 7 pm (the lights were turned off at other time), and the mice were allowed to access to water and feed freely. The experimental feed was mouse growth-stable feed (GB M2118), and the daily feeding and management of the animals were under the responsibility of the animal security department, which provided the animals with sufficient padding and fresh drinking water daily.

(29) Nomilin, deacetylnomilin, nomilin acid, deactylnomilin 17-β-D-glucopyranoside.

(30) Experimental grouping: male db/db mice (20±2 g) were selected, 18 male mice in each group were tested, and drinking bottles were sterilized weekly. The experimental groups included the normal control group (db/m, n=18), the model group (db/db, n=18), the nomilin group (db/db, n=18), the deacetylnomilin group (db/db, n=18), the nomilin acid group (db/db, n=18), the deactylnomilin 17-β-D-glucopyranoside group (db/db, n=18), the metformin group (db/db, n=18), the nomilin/metformin combination group (db/db, n=18), the deacetylnomilin/metformin combination group (db/db, n=18), the nomilin acid/metformin combination group (db/db, n=18), the deactylnomilin 17-β-D-glucopyranoside/combination group (db/db, n=18).

(31) Gavage doses: nomilin at a dose of 0.2 g/kg was gavaged per day for the nomilin group, deacetylnomilin at a dose of 0.2 g/kg was gavaged per day for the deacetylnomilin group, nomilin acid at a dose of 0.2 g/kg was gavaged per day for the nomilin acid group, deacetylnomilin 1743-D-glucopyranoside at a dose of 0.2 g/kg was gavaged per day for the deactylnomilin glucopyranoside group, metformin at a dose of 0.2 g/kg was gavaged per day for the metformin group, nomilin at a dose of 0.1 g/kg and metformin at a dose of 0.1 g/kg were simultaneously gavaged per day for the nomilin/metformin combination group, nomilin acid at a dose of 0.1 g/kg and metformin at a dose of 0.1 g/kg were simultaneously gavaged per day for the nomilin acid/metformin combination group, deacetylnomilin at a dose of 0.1 g/kg and metformin at a dose of 0.1 g/kg were simultaneously gavaged per day for the deacetylnomilin/metformin combination group, deactylnomilin 17-β-D-glucopyranoside at a dose of 0.1 g/kg and metformin at a dose of 0.1 g/kg were simultaneously gavaged per day for the deactylnomilin 17-β-D-glucopyranoside/metformin combination group, the gavage volume was 10 mL/kg, and the normal group and the model group were administrated with 10 mL/kg of distilled water. Two weeks later, the blood glucose values were measured by tail trimming method (Johnson's blood glucose meter) 1 h after the last administration, and serum insulin levels were measured with blood collected from orbital cavity by enzyme-linked immunosorbent assay (Elisa), and the average of each group was obtained. SPSS 16.0 software was used for statistical analysis. The data were expressed as mean and standard deviation. The data before and after were analyzed by t-test, and P<0.05 was considered statistically significant. The test results were shown in Table 4 below.

(32) TABLE-US-00004 TABLE 4 Blood glucose values and insulin of db/db mice after two weeks of daily gavage. Formulation and dose Blood glucose Insulin Group of administration value (mmol/L) (pg/ml) Normal control group None 6.0 ± 0.68 1.05 ± 0.39  Model group None 23.9 ± 4.2  10.56 ± 3.00   Metformin group Metformin 0.2 g/kg 12.2 ± 3.6** 8.19 ± 2.10** Nomilin group Nomilin acid 0.2 g/kg 10.1 ± 2 2** 8.82 ± 3.42** Nomilin acid group Nomilin acid 0.2 g/kg 11.0 ± 4.1*  8.08 ± 1 49** deacetylnomilin group deacetylnomilin 0.2 g/kg 11.9 ± 2 9** 8.59 ± 2.61** deactylnomilin 17-β-D- deactylnomilin 17-β-D- 12.8 ± 5.5** 8.40 ± 3.27** glucopyranoside group glucopyranoside 0.2 g/kg Nomilin/metformin Nomilin 0.1 g/kg +  6.2 ± 2.3** 4.16 ± 3.33** combination group metformin 0.1 g/kg Nomilin acid/metformin Nomilin acid 0.1 g/kg +  6.5 ± 3.5** 5.49 ± 1.98** combination group metformin 0.1 g/kg deacetylnomilin/metformin deacetylnomilin 0.1 g/kg +  6.6 ± 1.9** 5.83 ± 3.90  combination group metformin 0.1 g/kg deactylnomilin 17-β-D- deactylnomilin 17-β-D-  6.4 ± 1.2** 5.17 ± 1.04** glucopyranoside/metformin glucopyranoside 0.1 g/kg + combination group metformin 0.1 g/kg Note **after independent t-test, compared with the model group, the difference was extremely significant (P < 0.01) *after independent t-test, compared with the model group, the difference was extremely significant (P < 0.05)

(33) Discussion of Experimental Results

(34) From the above results, it could be seen that, compared with the model group, either in single administration or in combination administration with metformin, nomilin and derivatives thereof could significantly reduce the blood glucose levels in the db/db diabetic mice. When nomilin and derivatives thereof were administrated in combination with metformin, significantly improved effect was observed relative to the single administration thereof, showing a synergistic effect. In addition, when nomilin and derivatives thereof were administrated in combination with metformin, as compared with their single administration, the doses of both could be effectively reduced while comparable glucose-lowering effects could still be achieved, which improved the safety of therapeutic regimen and reduced side effects.

(35) Meanwhile, the limonoid compound represented by nomilin and derivatives thereof could significantly improve the sensitivity to insulin; and especially when administrated in combination with metformin, it could significantly improve the utilization efficiency of insulin in the body, improve the glucose metabolism of the body, and improve the functions relevant to the glucose metabolism in diabetes mice.

EXAMPLE 5

(36) Effects of a Limonoid Compound, Metformin or a Combination Thereof on Blood Glucose in a Mouse Model of Type II Diabetes with Pancreatic Islet Damage and Obesity

(37) In this example, a mouse model of type II diabetes with pancreatic islet damage and obesity was established by multiple modeling ICR mice with a small dose of streptozotocin (STZ), following with continuous high-fat diets (refer to the prior art literature: Zhang Jiyuan et al, Study on the effect of three plant extracts on improving glucose and lipid metabolism in type 2 diabetic mice, Food and Machinery, 2016, 32 (12): 142-147). The limonoid compound was selected from the group consisting of nomilin 17-β-D-glucopyranoside, deacetylnomilin 17-β-D-glucopyranoside, and nomilinic acid 17-β-D-glucopyranoside, and a metformin single administration group, nomilin 17-β-D-glucopyranoside single administration group, deacetylnomilin single administration group, nomilinic acid 17-β-D-glucopyranoside single administration group, and combination thereof with metformin administration groups were set, respectively.

(38) Conditions of experimental feeding: ICR mice (20±2 g), aged 6 weeks, purchased from Zhejiang Academy of Medical Sciences, and subjected to experimental feeding after 7 days of preliminary feeding. It should be noted that the conditions for raising the mice were as follows: the temperature was 23±1° C., the humidity was 55±10%, the lights were turned on between 7 am and 7 pm (the lights were turned off at other time), and the mice were allowed to access to water and feed freely. The experimental feed was mouse growth-stable feed (GB M2118), and the daily feeding and management of the animals were under the responsibility of the animal security department, which provided the animals with sufficient padding and fresh drinking water daily.

(39) Experimental grouping: 15 male mice were randomly selected as the normal control group, and the remaining mice were subjected to a high-fat diet (high-fat diet formula: cholesterol 1%, egg yolk powder 10%, lard oil 10%, and basic feed 79%, for establishing an obesity mouse model) for consecutive 4 weeks and intraperitoneal injection of STZ at a dose of 35 mg/kg for three consecutive days. After one week, the mice were subject to 24 hours of fasting and water deprivation, their fasting blood glucose was measured, and the mice with a blood glucose level of 15 to 25 mmol/L were selected and undifferentiatedly grouped and used in the experiment, continuously subjected to the high-fat diet, 15 mice in each group, and subjected to blood sampling and detection of indicators after 3 weeks of administration.

(40) Gavage doses: the gavage dose was 0.5 g/kg per day for the nomilin 17-β-D-glucopyranoside group, the gavage dose was 0.5 g/kg per day for the deacetylnomilin 17-β-D-glucopyranoside group, the gavage dose was 0.5 g/kg per day for the nomilinic acid 17-β-D-glucopyranoside group, metformin at a dose of 0.5 g/kg was gavaged per day for the metformin group, nomilin glucopyranoside at a dose of 0.25 g/kg and metformin at a dose of 0.25 g/kg were simultaneously gavaged per day for the nomilin 17-β-D-glucopyranoside/metformin combination group, deacetylnomilin 17-β-D-glucopyranoside at a dose of 0.25 g/kg and metformin at a dose of 0.25 g/kg were simultaneously gavaged per day for the deacetylnomilin glucopyranoside/metformin combination group, nomilinic acid 17-β-D-glucopyranoside at a dose of 0.25 g/kg and metformin at a dose of 0.25 g/kg were simultaneously gavaged per day for the nomilinic acid 17-β-D-glucopyranoside/metformin combination group, the gavage volume was 10 mL/kg, and the normal group and the model group were administrated with 10 mL/kg of distilled water. Two weeks later, the blood glucose values were measured by tail trimming method (Johnson's blood glucose meter) 1 h after the last administration, and the average of each group was obtained. SPSS 16.0 software was used for statistical analysis. The data were expressed as mean and standard deviation. The data before and after were analyzed by t-test, and P<0.05 was considered statistically significant. The test results were shown in Table 5 below.

(41) TABLE-US-00005 TABLE 5 Blood glucose values of STZ mice after three weeks of daily gavage. Blood glucose Formulation and dose value Group of administration (mmol/L) Normal control group None 5.9 ± 0.54 Model group None 29.6 ± 6.0  Metformin group Metformin 0.5 g/kg 17.9 ± 3.6** Nomilin 17-β-D- Nomilin 17-β-D- 12.8 ± 2.9** glucopyranoside glucopyranoside 0.5 g/kg group Deacetylnomilin Deacetylnomilin 17-β-D- 12.7 ± 2.5** 17-β-D- glucopyranoside 0.5 g/kg glucopyranoside group Nomilinic acid Nomilinic acid 17-β-D- 13.5 ± 2.4** 17-β-D- glucopyranoside 0.5 g/kg glucopyranoside group Nomilin 17-β-D- Nomilin 17-β-D-  5.8 ± 1.7** glucopyranoside/ glucopyranoside 0.25 g/kg + metformin combi- metformin 0.25 g/kg nation group Deacetylnomilin Deacetylnomilin 17-β-D-  6.3 ± 1.3** 17-β-D- glucopyranoside 0.25 g/kg + glucopyranoside/ metformin 0.25 g/kg metformin combi- nation group Nomilinic acid Nomilinic acid 17-β-D-  6.0 ± 2.0** 17-β-D- glucopyranoside 0.25 g/kg + glucopyranoside/ metformin 0.25 g/kg metformin combi- nation group Note **After independent t-test, compared with the model group, the difference was extremely significant (P < 0.01) * After independent t-test, compared with the model group, the difference was extremely significant (P < 0.05)

(42) Discussion of Experimental Results

(43) From the above results, it could be seen that, compared with the model group, either in single administration or in combination administration with metformin, the three limonoid glycosides all could significantly lower the blood glucose levels in the mice of the STZ type II diabetes model. When they were administrated in combination with metformin, their effects were significantly increased as compared with their single administration, similar to the normal mice in blood glucose level, showing a synergistic effect. In addition, when the above three limonoid glycosides were administration in combination with metformin, as compared with their single administration, the doses of both could be effectively reduced while comparable glucose-lowering effects could still be achieved, which improved the safety of therapeutic regimen and reduced side effects.

EXAMPLE 6

(44) Effects of a Limonoid Compound, Metformin or a Combination Thereof on Body Weight, Triglycerides, and Total Cholesterol in an Obesity Mouse Model

(45) In this example, ICR mice were used to complete the experiment for evaluation of lipid lowering and weight loss in animals (body weight, serum cholesterol, and serum triglycerides).

(46) In this example, the limonoid compound was nomilin, and a metformin single administration group, nomilin single administration group, and metformin/nomilin combination administration groups (dose A and B groups) were set, respectively.

(47) Conditions of experimental feeding: ICR mice, purchased from the Animal Center of Zhejiang Academy of Medical Sciences, half male and half female, body weight (20±2 g), fed with basic feed provided by the Animal Center of Zhejiang Medical Academy. It should be noted that the conditions for raising the mice were as follows: the temperature was 23±1° C., the humidity was 55±10%, the lights were turned on between 7 am and 7 pm (the lights were turned off at other time), and the mice were allowed to access to water and feed freely. The daily feeding and management of the animals were under the responsibility of the animal security department, which provided the animals with sufficient padding and fresh drinking water daily.

(48) The ICR mice were randomly divided into the normal group and the high-fat feed group, according to body weight. The normal group was fed with ordinary feed from the beginning to the end of the experiment. The rest of the animals were fed with a high-fat feed (high-fat diet formula: cholesterol 1%, egg yolk powder 10%, lard oil 10%, basic feed 79%, to establish a mouse model of obesity), body weight was measured after 3 weeks, in which the body weight of the animals in the high-fat feed group was higher than that in the normal control group, and t-test indicated that the data difference was significant and the modeling was successful (the data were not shown). The mice determined to have formed an obesity model were divided into a model control group (n=15), a nomilin control group (n=15), a metformin group (n=15), a of nomilin/metformin combination dose group A (n=15), and a nomiline/metformin combination dose group B (n=15).

(49) Gavage dose: nomilin at a dose of 0.1 g/kg was gavaged per day for the nomilin group, metformin at a dose of 0.1 g/kg was gavaged per day for the metformin group, nomilin at a dose of 0.05 g/kg and metformin at a dose of 0.05 g/kg were simultaneously gavaged per day for the combination dose group A, nomilin at a dose of 0.1 g/kg and metformin at a dose of 0.1 g/kg were simultaneously gavaged per day for the combination dose group B, the gavage volume was 10 mL/kg, and the normal group and the model group were administrated with 10 mL/kg of distilled water. Two weeks later, the mice were weighed, blood sampled from orbital cavity, measured to determine serum total cholesterol and triglyceride levels, and the average values of each group were obtained. SPSS 16.0 software was used for statistical analysis. The data were expressed as mean and standard deviation. The data before and after were analyzed by t-test, and P<0.05 was considered statistically significant. The test results were shown in Table 6 below.

(50) TABLE-US-00006 TABLE 6 Weight gaining rate, serum triglycerides, and total cholesterol values of ICR mice after two weeks of daily gavage. Body weight Total Formulation and dose gaining Triglycerides cholesterol Group of administration rate (%) (mmol/L) (mmol/L) Normal control group None 6.16 0.49 ± 0.19  1.67 ± 0.12 Model group None 13.45 1.35 ± 0.17  3.49 ± 0.31 Nomilin group Nomilin 0.1 g/kg 6.17 1.08 ± 0.33**  3.09 ± 0.16* Metformin group Metformin 0.1 g/kg 11.56 1.17 ± 0.30** 3.35 ± 0.07 Combination dose Nomilin 0.05 g/kg + 5.76 1.24 ± 0.28*   3.15 ± 0.14* group A metformin 0.05 g/kg Combination dose Nomilin 0.1 g/kg + 2.74 0.87 ± 0.46**  2.60 ± 0.20** group B metformin 0.1 g/kg Note **After independent t-test, compared with the model group, the difference was extremely significant (P < 0.01) *After independent t-test, compared with the model group, the difference was extremely significant (P < 0.05)

(51) Discussion of Experimental Results

(52) From the above results, it could be seen that, compared with the model group, the weight gaining rate of the combination dose group A and the high-dose group B were much lower than that of the model group, and their effect was superior to that of the single administration group, and the dose group B had better effect, and the two drugs showed a synergistic effect; when maintaining essentially comparable indicators such as body weight gaining rate, total serum cholesterol, and serum triglycerides, the combination dose group A effectively reduced the dose of positive control metformin, thereby reducing the side effects and increasing the safety of therapeutic regimen. Therefore, the combination administration of limonoid compounds and biguanide compounds could effectively improve the effect of lipid lowering and weight loss.

EXAMPLE 7

(53) Method for Preparing a Tablet Containing Combination Product of Nomilin and Metformin

(54) In this example, a method for preparing a tablet of a combination product (nomilin and metformin) of the present invention was exemplarily provided. A single tablet contained the following ingredients: 50 mg of nomilin, 400 mg of metformin hydrochloride, 20 mg of hydroxypropylmethylcellulose, 30 mg of sodium carboxymethylcellulose, and 20 mg of microcrystalline cellulose, 5.2 mg of magnesium stearate, 20.8 mg of Opadry, and there were a total of 1000 tablets.

(55) The preparation method comprised the following steps:

(56) a) dissolving 50 g of nomilin in 5 L of 50% ethanol;

(57) b) passing the raw and auxiliary materials through 100 mesh sieves, leaving them on standby;

(58) c) weighing 400 g of metformin hydrochloride, 20 g of hydroxypropylmethylcellulose, 30 g of sodium carboxymethylcellulose, and 20 g of microcrystalline cellulose, placing in a fluidized bed, and setting an inlet air volume of 500±50 m.sup.3/h, an inlet air temperature of 90±5° C., and a product temperature of 70±5° C., to perform hot melt granulation;

(59) d) spraying a nomilin solution into the fluidized bed, setting an atomizing pressure of 1.0±0.2 bar, and a spraying speed of 30±10 Hz, to perform one-step granulation;

(60) e) passing the resultant granules through a 1.0 mm round-hole screen to perform dry granulation;

(61) f) adding 5.2 g of magnesium stearate and mixing for 5 min;

(62) g) tabletting by using a 17×8.5 mm oval puncher at a pressure of 15 KN;

(63) h) dissolving 20.8 g of Opadry 85F32004 in distilled water at a ratio of 1:4, setting parameters of a coating pan as: bed temperature of 40±2° C., air temperature of 48±2° C., atomizing pressure of 0.6 Mpa, pan speed of 7 rpm, spray volume of 120 g/min, to complete film coating.