Composition for synergistically inhibiting pancreatic lipase and its application

Abstract

The present invention discloses a composition for synergistically inhibiting pancreatic lipase and its application, belonging to the technical field of natural medicines. The composition of the present invention is composed of isorhamnetin and compound A; wherein compound A is selected from EGCG or luteolin. The composition of the present invention can inhibit the activity of pancreatic lipase, thereby achieving the effect of preventing or treating obesity. There is a synergistic effect between the components in the composition. The combined use of the components can greatly reduce the dosage of each component when used alone, and improve the inhibitory activity of pancreatic lipase. Therefore, when achieving the corresponding effect of preventing or treating obesity, low-dose combined use can effectively reduce the occurrence of drug resistance in the body.

Description

Technical Field

[0001] The invention belongs to the technical field of natural medicines, and in particular relates to a composition for synergistically inhibiting pancreatic lipase and an application thereof. Background Art

[0002] The number of obese people in the world continues to rise, and the age of the onset population is beginning to be younger. This has become a global health problem. Many diseases, such as heart disease, diabetes, Alzheimer's disease, atherosclerosis and cancer, are closely related to obesity. Finding effective metabolic enzyme inhibitors is one of the effective methods for treating obesity caused by diet, because it can prevent the absorption and digestion of fat. Pancreatic lipase is a key enzyme for digesting dietary fat. Inhibiting pancreatic lipase activity to reduce the digestion and absorption of dietary triglycerides has become the focus of pharmacological intervention strategies in recent years. Some lipid-lowering drugs, such as orlistat, can inhibit pancreatic lipase in the gastrointestinal tract to prevent triglycerides from being hydrolyzed into free fatty acids and monoacylglycerides, thereby reducing the absorption of dietary triglycerides and promoting their excretion. However, continuous use can cause many adverse reactions, such as oily stools, abdominal discomfort, diarrhea, rectal discomfort, headache and fatigue, and even urinary tract infections. Therefore, screening for safer and more effective pancreatic lipase inhibitors from natural products has become a research hotspot for the prevention and treatment of obesity. At present, most of the research on pancreatic lipase inhibitors focuses on single compounds. Continuous use will produce certain side effects and tolerance, and there are few reports on the synergistic effect between active molecules. Therefore, the use of compound combination therapy to inhibit pancreatic lipase activity is of great significance for improving human obesity. Summary of the invention

[0003] The present invention provides a composition, which consists of isorhamnetin and compound A; wherein compound A is selected from EGCG or luteolin; when compound A is EGCG, the mass ratio of EGCG to isorhamnetin is selected from 20:50 to 30:30; when compound A is luteolin, the mass ratio of luteolin to isorhamnetin is selected from 8:50 to 10:30.

[0004] The above composition has an inhibitory effect on pancreatic lipase. Based on this, the present invention provides the use of the above composition in preparing a drug with a pancreatic lipase inhibitory effect.

[0005] Those skilled in the art know that pancreatic lipase is closely related to obesity. Based on this, the present invention provides the use of the above composition in the preparation of a drug for treating obesity.

[0006] In the above composition, when the mass ratio of EGCG and isorhamnetin is 20:30, there is a synergistic effect between the two in terms of pancreatic lipase inhibition; when the mass ratio of luteolin and isorhamnetin is 8:30, there is a synergistic effect between the two in terms of pancreatic lipase inhibition. Based on this, in a specific embodiment, the composition provided by the present invention is composed of EGCG and isorhamnetin in a mass ratio of 20:30; or, it is composed of luteolin and isorhamnetin in a mass ratio of 8:30.

[0007] According to the above content, a drug having a synergistic inhibitory effect on pancreatic lipase can be provided accordingly, the drug containing the above composition of the present invention. The drug contains a pharmaceutically acceptable carrier, solvent, diluent, excipient or other medium, and can be prepared into corresponding powders, granules, capsules, injections, oral liquids, tablets and other dosage forms according to different needs.

[0008] The beneficial effects of the present invention are:

[0009] The composition of the present invention can inhibit the activity of pancreatic lipase, thereby achieving the effect of preventing or treating obesity. There is a synergistic effect between the components in the composition. The combined use of the components can greatly reduce the dosage of each component when used alone, and improve the inhibitory activity on pancreatic lipase. Therefore, when achieving the corresponding effect of preventing or treating obesity, the low-dose combined use can effectively reduce the occurrence of drug resistance in the body. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 is the inhibition curve of EGCG, isorhamnetin and their combination (20:30) on pancreatic lipase;

[0011] Figure 2 The Fa-CI trend diagram of the combination of EGCG and isorhamnetin (20:30) on pancreatic lipase;

[0012] Figure 3 is the inhibition curve of luteolin, isorhamnetin and their combination (8:30) on pancreatic lipase;

[0013] Figure 4 This is the Fa-CI trend diagram of the combination of luteolin and isorhamnetin (8:30) on pancreatic lipase. DETAILED DESCRIPTION

[0014] EGCG, molecular formula is C 22 H 18 O 11 ; Molecular weight: 458.38; CAS registration number: 989-51-5, structural formula:

[0015]

[0016] Isorhamnetin, molecular formula: C 16 H 12 O7; molecular weight: 316.26; CAS registration number: 480-19-3, structural formula:

[0017]

[0018] Luteolin, molecular formula is C 15 H 10 O6; molecular weight: 286.24; CAS registration number: 491-70-3, structural formula:

[0019]

[0020] Other materials used in the present invention, if not otherwise stated, can be obtained through commercial channels. Other terms used in the present invention, unless otherwise specified, generally have the meanings commonly understood by those of ordinary skill in the art. The present invention is further described in detail below in conjunction with specific examples and with reference to data. The following examples are only for illustrating the present invention, and are not intended to limit the scope of the present invention in any way.

[0021] Example 1

[0022] The in vitro inhibition test of pancreatic lipase activity is as follows:

[0023] The test object was dissolved in dimethyl sulfoxide (DMSO) to prepare a 10 mg / mL stock solution, and then the stock solution was diluted with PBS (after dilution, the DMSO content was less than 5%), and a concentration gradient was set to form a sample to be tested. A 1 mg / mL pancreatic lipase solution (centrifuged to obtain the supernatant) and a 0.1 mM substrate 4-Methylumbelliferyloleate (4-MUO) solution were prepared with PBS buffer.

[0024] Add 25 μL of the sample to be tested and 25 μL of pancreatic lipase (1 mg / mL) to each well, mix well, add 50 μL of reaction substrate 4-MUO, incubate at 25°C for 20 min, then add 100 μL of sodium citrate buffer solution, and measure the fluorescence value of each well with an enzyme-labeled instrument. The excitation wavelength is 320 nm and the emission wavelength is 450 nm.

[0025] The inhibition rate of pancreatic lipase activity at each gradient was calculated according to the FL value:

[0026] Inhibition rate = [1-(FL 样品 –FL 样品空白 ) / (FL 阴性对照 -FL空白 )]×100%

[0027] Among them, FL 样品 Refers to the fluorescence value of the sample group, FL 样品空白 Refers to the fluorescence value of the sample blank group, FL 阴性对照 Refers to the fluorescence value of the negative control group, FL 空白 Refers to the fluorescence value of the blank group.

[0028] According to the inhibition rate of pancreatic lipase activity at each gradient, the half inhibitory concentration (IC 50 The calculation and statistical process were performed using SPSS 20.0.

[0029] In the above operation steps, specifically, each group is represented as:

[0030] Sample group: 25 μL of sample to be tested + 25 μL of enzyme;

[0031] Sample blank group: 25 μL of the sample to be tested + 25 μL PBS;

[0032] Negative control group: 25 μL PBS + 25 μL enzyme;

[0033] Blank group: 50 μL PBS.

[0034] The positive control was orlistat.

[0035] The experimental instruments and reagents included: pancreatic lipase (Type II, L3126) and 4-Methylumbelliferyl oleate (4-MUO, S75164) purchased from Sigma; EGCG, isorhamnetin and luteolin (Beijing Solebao); MilliporeSimplicity water purification system (Millipore, France), and microplate reader TECAN infinite M200 PRO (Teacan Group Ltd., Swizerland).

[0036] Specifically, EGCG, isorhamnetin and luteolin were used as test objects, and the inhibitory activity of each test object on pancreatic lipase was tested using the above method. The half inhibitory concentration IC 50 Value representation.

[0037] The concentration gradient settings of each test object were as follows: EGCG: 30μg / mL, 15μg / mL, 7.5μg / mL, 3.75μg / mL; Isorhamnetin: 60μg / mL, 30μg / mL, 15μg / mL, 7.5μg / mL; Luteolin: 20μg / mL, 10μg / mL, 5μg / mL, 2.5μg / mL; Orlistat: 0.2μg / mL, 0.1μg / mL, 0.05μg / mL, 0.025μg / mL.

[0038] The test results are shown in Table 1 below.

[0039] Table 1

[0040]

[0041] The data are derived from the results of three independent experiments and are expressed as “mean ± SD”.

[0042] Example 2

[0043] The combination of EGCG and isorhamnetin was used as the test object, and the inhibitory activity of each test object on pancreatic lipase was tested using the method in Example 1. The half inhibitory concentration IC 50 Value representation.

[0044] The specific test objects are: EGCG and isorhamnetin (20:50), EGCG and isorhamnetin (20:30), EGCG and isorhamnetin (30:30). The above ratios are all mass ratios.

[0045] The concentration gradient of each test object was set as follows:

[0046] EGCG and isorhamnetin (20:50): (20+50) μg / mL, (10+25) μg / mL, (5+12.5) μg / mL, (2.5+6.25) μg / mL;

[0047] EGCG and isorhamnetin (20:30): (20+30) μg / mL, (10+15) μg / mL, (5+7.5) μg / mL, (2.5+3.75) μg / mL;

[0048] EGCG and isorhamnetin (30:30): (30+30) μg / mL, (15+15) μg / mL, (7.5+7.5) μg / mL, (3.75+3.75) μg / mL.

[0049] Concentration gradient setting principle: Taking EGCG and isorhamnetin (20:30) as an example, the initial concentration of the composition is set to 50 μg / mL, that is, the initial concentration of EGCG in the sample solution of the composition is 20 μg / mL, and the initial concentration of isorhamnetin is

[0050] 30μg / mL. Based on the initial concentration of 50μg / mL, the sample solution of the composition was diluted 2 times in sequence, namely 50μg / mL, 25μg / mL, 12.5μg / mL, and 6.25μg / mL. Similarly, in the composition of EGCG and isorhamnetin (20:50), the initial concentration of the composition was set to 70μg / mL, and it was diluted 2 times in sequence to form a concentration gradient. Other compositions also follow this principle.

[0051] The test results are shown in Table 2.

[0052] Table 2

[0053]

[0054] The data are derived from the results of three independent experiments and are expressed as “mean ± SD”.

[0055] It can be seen from Tables 1 and 2 that the combined use of EGCG and isorhamnetin, when the mass ratio of the two is 20:30, can significantly reduce the dosage of each component when used alone and improve the inhibitory activity against pancreatic lipase while achieving a similar inhibition rate.

[0056] Example 3

[0057] The combination of luteolin and isorhamnetin was used as the test object, and the inhibitory activity of each test object on pancreatic lipase was tested using the method in Example 1. The half inhibitory concentration IC 50 Value representation.

[0058] The specific test objects are: luteolin and isorhamnetin (8:50), luteolin and isorhamnetin (8:30), luteolin and isorhamnetin (10:30). The above ratios are all mass concentration ratios.

[0059] The concentration gradient of each test object was set as follows:

[0060] Luteolin and isorhamnetin (8:50): (8+50) μg / mL, (4+25) μg / mL, (2+12.5) μg / mL, (1+6.25) μg / mL;

[0061] Luteolin and isorhamnetin (8:30): (8+30) μg / mL, (4+15) μg / mL, (2+7.5) μg / mL, (1+3.75) μg / mL;

[0062] Luteolin and isorhamnetin (10:30): (10+30)μg / mL, (5+15)μg / mL, (2.5+7.5)μg / mL, (1.25+3.75)μg / mL.

[0063] Principles for setting concentration gradient: Taking luteolin and isorhamnetin (8:30) as an example, the initial concentration of the composition is set to 38 μg / mL, that is, the initial concentration of luteolin in the composition sample solution is 8 μg / mL, and the initial concentration of isorhamnetin is 30 μg / mL. On the basis of the initial concentration of 38 μg / mL, the composition sample solution is diluted 2 times in sequence, namely 38 μg / mL, 19 μg / mL, 9.5 μg / mL, and 4.75 μg / mL. Similarly, in the composition of luteolin and isorhamnetin (10:30), the initial concentration of the composition is set to 40 μg / mL, and it is diluted 2 times in sequence to form a concentration gradient. Other compositions also follow this principle.

[0064] The test results are shown in Table 3.

[0065] Table 3

[0066]

[0067] The data are derived from the results of three independent experiments and are expressed as “mean ± SD”.

[0068] It can be seen from Tables 1 and 3 that the combined use of luteolin and isorhamnetin, when the mass ratio of the two is 8:30, can significantly reduce the dosage of each component when used alone and improve the inhibitory activity against pancreatic lipase when a similar inhibition rate is achieved.

[0069] Determination of drug combination coefficient

[0070] The combination coefficient (CI) of the compositions in Example 2 and Example 3 was measured, the CI value was calculated using the software CompuSyn, and the synergistic effect between the drugs was evaluated.

[0071] The combination coefficient of the composition depends on the inhibition rate of the composition at different concentration gradients, as well as the inhibition rate of each monomer compound in the composition at different concentration gradients. Therefore, when determining the combination coefficient of the composition, it is necessary to set the concentration gradient of each monomer compound in the composition to obtain the corresponding inhibition rate.

[0072] Taking EGCG and isorhamnetin (20:30) in Example 2 as an example, the initial concentration of the composition is set to 50 μg / mL, the initial concentration of EGCG is 20 μg / mL, and the initial concentration of isorhamnetin is 30 μg / mL; therefore, the gradient concentration setting of EGCG is based on the initial concentration of 20 μg / mL, and is diluted 2 times in sequence, which are 20 μg / mL, 10 μg / mL, 5 μg / mL, and 2.5 μg / mL in sequence; the gradient concentration setting of isorhamnetin is based on the initial concentration of 30 μg / mL, and is diluted 2 times in sequence, which are 30 μg / mL, 15 μg / mL, 7.5 μg / mL, and 3.75 μg / mL in sequence; the gradient concentration setting of the composition of EGCG and isorhamnetin (20:30) is based on the initial concentration of 50 μg / mL, and is diluted 2 times in sequence, which are 50 μg / mL, 25 μg / mL, 12.5 μg / mL, and 6.25 μg / mL in sequence.

[0073] The inhibition rates of EGCG, isorhamnetin, and EGCG and isorhamnetin (20:30) at different concentration gradients were statistically analyzed. Figure 1 As shown. The inhibition rates of EGCG under the concentration gradient were 9.5±1.3%, 15.5±1.2%, 28.5±1.1%, 45.2±2.2% (from low concentration to high concentration), the inhibition rates of isorhamnetin under the concentration gradient were 15.6±1.1%, 28.5±1.1%, 38.2±1.2%, 48.1±2.3% (from low concentration to high concentration), and the inhibition rates of the EGCG and isorhamnetin (20:30) composition under the concentration gradient were 26.4±1.2%, 40.3±1.3%, 56.4±2.1%, 70.5±3.2% (from low concentration to high concentration). The above data were processed using CompuSyn software to obtain the CI values ​​of EGCG and isorhamnetin in the EGCG and isorhamnetin (20:30) composition, as shown in Table 4. Thus, it can be concluded that the Fa-CI trend of the combination of EGCG and isorhamnetin (20:30) in inhibiting pancreatic lipase, such as Figure 2 shown.

[0074] Taking luteolin and isorhamnetin (8:30) in Example 3 as an example, the initial concentration of the composition is set to 38 μg / mL, the initial concentration of luteolin is 8 μg / mL, and the initial concentration of isorhamnetin is 30 μg / mL; therefore, the gradient concentration setting of luteolin is based on the initial concentration of 8 μg / mL, and then diluted 2 times in sequence, which are 8 μg / mL, 4 μg / mL, 2 μg / mL, and 1 μg / mL in sequence; the gradient concentration setting of isorhamnetin is based on the initial concentration of 30 μg / mL, and then diluted 2 times in sequence, which are 30 μg / mL, 15 μg / mL, 7.5 μg / mL, and 3.75 μg / mL in sequence; the gradient concentration setting of the composition of luteolin and isorhamnetin (8:30) is based on the initial concentration of 38 μg / mL, and then diluted 2 times in sequence, which are 38 μg / mL, 19 μg / mL, 9.5 μg / mL, and 4.75 μg / mL in sequence.

[0075] The inhibition rates of luteolin, isorhamnetin, luteolin and isorhamnetin (8:30) at different concentration gradients were statistically analyzed. Figure 3 As shown. The inhibition rates of luteolin under the concentration gradient were 11.9±1.5%, 22.5±1.3%, 36.5±1.5%, 50.2±2.4% (from low concentration to high concentration), the inhibition rates of isorhamnetin under the concentration gradient were 15.6±1.1%, 28.5±1.1%, 38.2±1.2%, 48.1±2.3% (from low concentration to high concentration), and the inhibition rates of the combination of luteolin and isorhamnetin (8:30) under the concentration gradient were 20.4±1.2%, 36.3±2.1%, 48.4±2.3%, 60.5±3.8% (from low concentration to high concentration). The above data were processed by CompuSyn software to obtain the CI values ​​of luteolin and isorhamnetin in the combination of luteolin and isorhamnetin (8:30), as shown in Table 4. Thus, it can be concluded that the Fa-CI trend of the combination of luteolin and isorhamnetin (8:30) in inhibiting pancreatic lipase is as follows: Figure 4 shown.

[0076] Similarly, for other compositions, including EGCG and isorhamnetin (20:50), EGCG and isorhamnetin (30:30), luteolin and isorhamnetin (8:50), and luteolin and isorhamnetin (10:30), the concentration gradient setting of each monomer compound and the calculation of CI values ​​were all based on the above principles.

[0077] The measurement results are shown in Table 4:

[0078] Table 4

[0079]

[0080] The data are derived from the results of three independent experiments and are expressed as “mean ± SD”.

[0081] From Table 4, we can see that:

[0082] When EGCG and isorhamnetin were used together, the combined medication coefficient CI was about 1 when the mass ratio of the two was 20:50, showing an additive effect. When the mass ratio of the two was 20:30, the combined medication coefficient CI was less than 0.8, showing a strong synergistic effect. The mean combined medication index (CI avg ) is 0.66. When the mass ratio of the two is 30:30, the mean value of the combined drug index (CI avg ) was 0.92, showing a weak synergistic effect.

[0083] When the mass ratio of luteolin and isorhamnetin was 8:50, the combined medication coefficient CI was greater than 1, showing an antagonistic effect. 75 and GI 90 Both were less than 0.8, indicating that the two showed a strong synergistic effect when the mass ratio was 8:30. The mean value of the combined drug index (CI avg ) is 0.64. When the mass ratio of the two is 10:30, the combined drug coefficient CI is greater than 1, showing an antagonistic effect.

[0084] Comparative Example

[0085] In addition to the above implementation cases, the present invention also provides other cases that cannot form a synergistic effect with isorhamnetin during the combination screening process. In these cases, compound A involves kaempferol, quercetin, morin and phloretin. These cases are only part of the workload of the present invention at the time of creation. As shown below:

[0086] First, the IC values ​​of each monomer compound on pancreatic lipase were tested according to the method in the above example. 50 Values ​​are shown in Table 5 below:

[0087] Table 5

[0088]

[0089] The data are derived from the results of three independent experiments and are expressed as “mean ± SD”.

[0090] Then, take the IC of each monomer compound 50 The pancreatic lipase inhibition rate was determined by comparing the values ​​of the values, as shown in Table 6:

[0091] Table 6

[0092] Compound Mass concentration (μg / mL) Inhibition rate Isorhamnetin 30 48.1±2.3％ Kaempferol 50 53.1±1.8％ Quercetin 2 43.1±1.3％ Morin 4 52.7±3.1％ Phloretin 50 50.2±3.6％

[0093] The above monomer compounds were combined with isorhamnetin to measure the pancreatic lipase inhibition rate of the composition, as shown in the following Table 7:

[0094] Table 7

[0095]

[0096]

[0097] It can be seen from Table 7 that after the above monomer compounds are combined with isorhamnetin, the inhibition rate of the combination is directly lower than the inhibition rate of the single compound. The combination actually exhibits an antagonistic effect without a synergistic effect.

[0098] The above is only a preferred embodiment of the present invention, and does not limit the present invention in other forms. Any technician familiar with the profession may use the above disclosed technical content to change or modify it into an equivalent embodiment with equivalent changes. However, any simple modification, equivalent change and modification made to the above embodiment according to the technical essence of the present invention without departing from the technical solution of the present invention still belongs to the protection scope of the technical solution of the present invention.

Claims

1. A composition for synergistically inhibiting pancreatic lipase, characterized in that: Composed of isorhamnetin and compound A; wherein compound A is selected from EGCG or luteolin; When compound A is EGCG, the mass ratio of EGCG to isorhamnetin is 20:30; when compound A is luteolin, the mass ratio of luteolin to isorhamnetin is 8:

30.

2. Use of the composition for synergistically inhibiting pancreatic lipase according to claim 1 in the preparation of a drug having a synergistic inhibitory effect on pancreatic lipase.

3. Use of the composition for synergistically inhibiting pancreatic lipase according to claim 1 in the preparation of a drug for treating obesity.

4. A drug having a synergistic inhibitory effect on pancreatic lipase, characterized in that: The active ingredient of the medicine is the composition for synergistically inhibiting pancreatic lipase as claimed in claim 1.

5. The drug having a synergistic inhibitory effect on pancreatic lipase according to claim 4, characterized in that: A pharmaceutically acceptable carrier is further added.

6. The drug having a synergistic inhibitory effect on pancreatic lipase according to claim 5, characterized in that: The dosage form of the drug is selected from powder, granules, capsules, injections, oral liquids or tablets.

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