Orlistat combination compositions and uses thereof

Abstract

The application discloses an orlistat combined composition and application thereof, and belongs to the technical field of natural medicines. The composition is composed of orlistat and a compound A; wherein the compound A is selected from luteolin, quercetin, dihydroquercetin, 3-O-methyl quercetin or EGCG. The composition can inhibit the activity of pancreatic lipase, thereby achieving the effect of treating obesity. The components in the composition have a synergistic effect. The combination of the components can greatly reduce the dosage of each component when used alone, reduce the toxic and side effects of the drugs, and achieve a synergistic treatment effect. Therefore, when the corresponding effect of treating obesity is achieved, the combination of the drugs can effectively reduce the occurrence of drug resistance of the body, and has a good medical application prospect.

Description

Technical Field

[0001] This invention belongs to the field of natural medicine technology, specifically relating to orlistat combination compositions and their applications. Background Technology

[0002] In recent years, with changes in modern lifestyles and dietary structures, the incidence and mortality rates of obesity and obesity-related metabolic diseases have been rising year by year, becoming an increasingly serious global public health problem. Although genetic, physiological, medical, and behavioral factors are all causes of obesity, the most significant cause is the long-term imbalance between energy intake and energy expenditure. Currently, various strategies are used to treat overweight and obesity, including regulating lipid metabolism, inhibiting nutrient absorption, regulating fat signaling transduction, and regulating satiety. One of the most promising strategies is the development of inhibitors of nutrient digestion and absorption, attempting to control overweight and obesity by reducing energy intake in the gastrointestinal tract without altering any central mechanisms. Pancreatic lipase, a key digestive enzyme responsible for the hydrolysis of dietary triglycerides in the gastrointestinal tract, has become an important anti-obesity target and has attracted considerable attention. To date, orlistat is a weight-loss drug marketed as an irreversible pancreatic lipase inhibitor that directly targets serine residues in the mammalian pancreatic lipase catalytic triplet by forming a covalent bond. Although orlistat has excellent weight-loss effects, its numerous serious side effects (including urgency, frequent bowel movements, oily stools, diarrhea, and liver and kidney damage) severely hinder its long-term clinical use. Therefore, combining orlistat with other drugs to inhibit pancreatic lipase activity, producing a synergistic effect and relatively reducing the dosage, is of great significance for improving human obesity. Summary of the Invention

[0003] The present invention provides an orlistat composition comprising orlistat and compound A; wherein, compound A is selected from luteolin, quercetin, dihydroquercetin, 3-O-methylquercetin or EGCG;

[0004] When compound A is luteolin, the mass ratio of orlistat to luteolin is 0.1:10 to 0.2:8; when compound A is quercetin, the mass ratio of orlistat to quercetin is 0.1:4 to 0.2:2; when compound A is dihydroquercetin, the mass ratio of orlistat to dihydroquercetin is 0.1:70 to 0.2:60; when compound A is 3-O-methylquercetin, the mass ratio of orlistat to 3-O-methylquercetin is 0.1:60 to 0.2:50; when compound A is EGCG, the mass ratio of orlistat to EGCG is 0.1:30 to 0.2:20.

[0005] The above-mentioned orlistat composition has an inhibitory effect on pancreatic lipase. Based on this, the present invention provides the use of the above-mentioned orlistat composition in the preparation of a drug with pancreatic lipase inhibitory effect.

[0006] Those skilled in the art will know that pancreatic lipase is closely related to obesity. Based on this, the present invention provides the use of the above-mentioned orlistat composition in the preparation of a medicament for treating obesity.

[0007] In the above-mentioned orlistat compositions, a synergistic effect exists between orlistat and luteolin in the inhibition of pancreatic lipase when the mass ratio is 0.1:10 to 0.2:8; between orlistat and quercetin in the inhibition of pancreatic lipase when the mass ratio is 0.1:2 to 0.2:2; between orlistat and dihydroquercetin in the inhibition of pancreatic lipase when the mass ratio is 0.1:70 to 0.2:60; between orlistat and 3-O-methylquercetin in the inhibition of pancreatic lipase when the mass ratio is 0.1:60 to 0.2:50; and between orlistat and EGCG in the inhibition of pancreatic lipase when the mass ratio is 0.1:30 to 0.2:20. Based on this, in specific embodiments, the orlistat composition provided by the present invention is composed of orlistat and luteolin in a mass ratio of 0.1:10 to 0.2:8; or, composed of orlistat and quercetin in a mass ratio of 0.1:2 to 0.2:2; or, composed of orlistat and dihydroquercetin in a mass ratio of 0.1:70 to 0.2:60; or, composed of orlistat and 3-O-methylquercetin in a mass ratio of 0.1:60 to 0.2:50; or, composed of orlistat and EGCG in a mass ratio of 0.1:30 to 0.2:20.

[0008] In one specific embodiment, the orlistat composition of the present invention is composed of orlistat and luteolin in a mass ratio of 0.1:8; or, orlistat and quercetin in a mass ratio of 0.1:2; or, orlistat and dihydroquercetin in a mass ratio of 0.1:60; or, orlistat and 3-O-methylquercetin in a mass ratio of 0.1:50; or, orlistat and EGCG in a mass ratio of 0.1:20.

[0009] Based on the above, a drug with synergistic inhibitory effect on pancreatic lipase can be provided, comprising the above-mentioned orlistat composition. The drug includes pharmaceutically acceptable carriers, solvents, diluents, excipients, or other media, and can be prepared into various dosage forms such as powders, granules, capsules, injections, oral liquids, and tablets as required.

[0010] The beneficial effects of this invention are as follows:

[0011] The composition of this invention can inhibit the activity of pancreatic lipase, thereby achieving the effect of treating obesity. There is a synergistic effect among the components of the composition. Combined use of these components can significantly reduce the dosage of each component when used alone, reduce drug toxicity and side effects, and achieve a synergistic therapeutic effect. Therefore, when achieving the corresponding therapeutic effect on obesity, combined use can effectively reduce the occurrence of drug resistance in the body, and has good prospects for medical application. Attached Figure Description

[0012] Figure 1 Fa-CI trend graph of pancreatic lipase for the combination of orlistat and luteolin (0.1:8);

[0013] Figure 2 Fa-CI trend graph of pancreatic lipase for the combination of orlistat and quercetin (0.1:2);

[0014] Figure 3 Fa-CI trend graph of pancreatic lipase by the combination of orlistat and dihydroquercetin (0.1:60);

[0015] Figure 4 Fa-CI trend graph of pancreatic lipase by the combination of orlistat and 3-O-methylquercetin (0.1:50);

[0016] Figure 5 Fa-CI trend of pancreatic lipase in the combination of orlistat and EGCG (0.1:20). Detailed Implementation

[0017] Orlistat, molecular formula C 29 H 53 NO5; Molecular weight: 495.7; CAS Registry Number: 96829-58-2; Structural formula:

[0018]

[0019] Luteolin, with the molecular formula C 15 H 10 O6; Molecular weight: 286.24; CAS Registry Number: 491-70-3, Structural Formula:

[0020]

[0021] Quercetin, with the molecular formula C 15 H 10 O7; Molecular weight: 302.236; CAS Registry Number: 117-39-5; Structural formula:

[0022]

[0023] Dihydroquercetin (Taxifolin), with the molecular formula C2... 15 H 12 O7; Molecular weight: 304.25; CAS Registry Number: 480-18-2; Structural formula:

[0024]

[0025] 3-O-methylquercetin, with the molecular formula C 16 H 12 O7; Molecular weight: 316.262; CAS Registry Number: 1486-70-0; Structural formula:

[0026]

[0027] EGCG, with the molecular formula C 22 H 18 O 11 Molecular weight: 458.38; CAS Registry Number: 989-51-5; Structural formula:

[0028]

[0029] Other materials used in this invention, unless otherwise stated, are commercially available. Other terms used in this invention, unless otherwise specified, generally have the meanings commonly understood by those skilled in the art. The invention is further described in detail below with reference to specific embodiments and data. The following embodiments are merely illustrative and not intended to limit the scope of the invention in any way.

[0030] Example 1

[0031] The in vitro inhibition assay for pancreatic lipase activity is performed as follows:

[0032] The test substance was dissolved in dimethyl sulfoxide (DMSO) to prepare a 10 mg / mL stock solution. The stock solution was then diluted with PBS (after dilution, the DMSO content was less than 5%) to form a concentration gradient and prepare the test sample. A 1 mg / mL pancreatic lipase solution (centrifuged and the supernatant was collected) and a 0.1 mM substrate 4-methylumbelliferyloleate (4-MUO) solution were prepared using PBS buffer.

[0033] Add 25 μL of the sample to be tested and 25 μL of pancreatic lipase (1 mg / mL) to each well. After mixing the samples, add 50 μL of the reaction substrate 4-MUO and incubate at 25 °C for 20 min. Then add 100 μL of sodium citrate buffer solution. Measure the fluorescence value of each well using a microplate reader. The excitation wavelength is 320 nm and the emission wavelength is 450 nm.

[0034] Calculate the pancreatic lipase activity inhibition rate at each gradient based on the FL value:

[0035] Inhibition rate = [1-(FL)] 样品 –FL 样品空白 ) / (FL 阴性对照 -FL 空白 )]×100%

[0036] Among them, FL 样品 This refers to the fluorescence value of the sample group, FL. 样品空白 This refers to the fluorescence value of the blank sample group, FL. 阴性对照 This refers to the fluorescence value of the negative control group, FL. 空白 This refers to the fluorescence value of the blank control group.

[0037] Calculate the half-inhibition concentration (IC50) of the test subjects based on the inhibition rate of pancreatic lipase activity at each gradient. 50 The values ​​were calculated and statistically analyzed using SPSS 20.0.

[0038] Specifically, in the above operation steps, each group is represented as follows:

[0039] Sample set: 25 μL of test sample + 25 μL of enzyme;

[0040] Sample blank group: 25 μL of test sample + 25 μL of PBS;

[0041] Negative control group: 25 μL PBS + 25 μL enzyme;

[0042] Control group: 50 μL PBS.

[0043] The experimental instruments and reagents included: pancreatic lipase (Type II, L3126), 4-Methylumbelliferyl oleate (4-MUO, S75164), and orlistat, purchased from Sigma; luteolin, quercetin, dihydroquercetin, 3-O-methylquercetin, and EGCG (Beijing Solarbio); Millipore Simplicity water purification system (Millipore, France); and a TECAN infinite M200 PRO microplate reader (Teacan Group Ltd., Swizerland).

[0044] Specifically, orlistat, luteolin, quercetin, dihydroquercetin, 3-O-methylquercetin, and EGCG were used as test subjects. The above method was used to test the inhibitory activity of each test subject on pancreatic lipase, and the half-inhibitory concentration (IC50) was used as the target. 50 Value representation.

[0045] The concentration gradients for each test subject were set as follows: Orlistat: 0.2 μg / mL, 0.1 μg / mL, 0.05 μg / mL, 0.025 μg / mL; Luteolin: 20 μg / mL, 10 μg / mL, 5 μg / mL, 2.5 μg / mL; Quercetin: 4 μg / mL, 2 μg / mL, 1 μg / mL, 0.5 μg / mL; Dihydroquercetin: 70 μg / mL, 35 μg / mL, 17.5 μg / mL, 8.75 μg / mL; 3-O-methylquercetin: 60 μg / mL, 30 μg / mL, 15 μg / mL, 7.5 μg / mL; EGCG: 30 μg / mL, 15 μg / mL, 7.5 μg / mL, 3.75 μg / mL.

[0046] The experimental results are shown in Table 1 below.

[0047] Table 1

[0048]

[0049]

[0050] The data are derived from the results of three independent experiments and are expressed as "mean ± standard deviation".

[0051] Example 2

[0052] Using orlistat and luteolin as test subjects, the inhibitory activity of each test subject against pancreatic lipase was tested using the method described in Example 1, with the half-inhibitory concentration (IC50) as the criterion. 50 Value representation.

[0053] The specific ratios for each test subject were: orlistat and luteolin (0.1:10), orlistat and luteolin (0.1:8), and orlistat and luteolin (0.2:8). All ratios are by mass.

[0054] The concentration gradients for each test subject are set as follows:

[0055] Orlistat and luteolin (0.1:10): (0.1+10) μg / mL, (0.05+5) μg / mL, (0.025+2.5) μg / mL, (0.0125+1.25) μg / mL;

[0056] Orlistat and luteolin (0.1:8): (0.1+8) μg / mL, (0.05+4) μg / mL, (0.025+2) μg / mL, (0.0125+1) μg / mL;

[0057] Orlistat and luteolin (0.2:8): (0.2+8)μg / mL, (0.1+4)μg / mL, (0.05+2)μg / mL, (0.025+1)μg / mL.

[0058] Concentration gradient setting principle: Taking orlistat and luteolin (0.1:8) as an example, the initial concentration of the composition is set to 8.1 μg / mL, that is, the initial concentration of orlistat in the composition sample solution is 0.1 μg / mL, and the initial concentration of luteolin is 8 μg / mL. Based on the initial concentration of 8.1 μg / mL, the composition sample solution is successively diluted 2-fold to 8.1 μg / mL, 4.05 μg / mL, 2.025 μg / mL, and 1.0125 μg / mL. Similarly, in the orlistat and luteolin (0.1:10) composition, the initial concentration is set to 10.1 μg / mL, and it is successively diluted 2-fold to form a concentration gradient. Other compositions also follow this principle.

[0059] The experimental results are shown in Table 2.

[0060] Table 2

[0061]

[0062]

[0063] The data are derived from the results of three independent experiments and are expressed as "mean ± standard deviation".

[0064] Tables 1 and 2 show that the combined use of orlistat and luteolin, while achieving similar inhibition rates, significantly reduces the dosage of each component compared to their individual use, and enhances their inhibitory activity against pancreatic lipase. The effect is particularly pronounced when the mass ratio of the two is 0.1:8.

[0065] Example 3

[0066] Using a combination of orlistat and quercetin as the test subject, the inhibitory activity of each test subject against pancreatic lipase was tested using the method described in Example 1, with the half-inhibitory concentration (IC50) as the criterion. 50 Value representation.

[0067] The specific ratios for each test subject were: orlistat and quercetin (0.1:4), orlistat and quercetin (0.1:2), and orlistat and quercetin (0.2:2). All ratios are by mass.

[0068] The concentration gradients for each test subject are set as follows:

[0069] Orlistat and quercetin (0.1:4): (0.1+4) μg / mL, (0.05+2) μg / mL, (0.025+1) μg / mL, (0.0125+0.5) μg / mL;

[0070] Orlistat and quercetin (0.1:2): (0.1+2) μg / mL, (0.05+1) μg / mL, (0.025+0.5) μg / mL, (0.0125+0.25) μg / mL;

[0071] Orlistat and quercetin (0.2:2): (0.2+2)μg / mL, (0.1+1)μg / mL, (0.05+0.5)μg / mL, (0.025+0.25)μg / mL.

[0072] Concentration gradient setting principle: Taking orlistat and quercetin (0.1:2) as an example, the initial concentration of the composition is set to 2.1 μg / mL, that is, the initial concentration of orlistat in the composition sample solution is 0.1 μg / mL, and the initial concentration of quercetin is 2 μg / mL. Based on the initial concentration of 2.1 μg / mL, the composition sample solution is successively diluted 2-fold to 2.1 μg / mL, 1.05 μg / mL, 0.525 μg / mL, and 0.2625 μg / mL. Similarly, in the orlistat and quercetin (0.1:4) composition, the initial concentration is set to 4.1 μg / mL, and it is successively diluted 2-fold to form a concentration gradient. Other compositions also follow this principle.

[0073] The experimental results are shown in Table 3.

[0074] Table 3

[0075]

[0076]

[0077] The data are derived from the results of three independent experiments and are expressed as "mean ± standard deviation".

[0078] Tables 1 and 3 show that the combined use of orlistat and quercetin, while achieving similar inhibition rates, significantly reduces the dosage of each component compared to their individual use, and enhances their inhibitory activity against pancreatic lipase. The effect is particularly pronounced when the mass ratio of the two is 0.1:2.

[0079] Example 4

[0080] Using a combination of orlistat and dihydroquercetin as the test subject, the inhibitory activity of each test subject against pancreatic lipase was tested using the method described in Example 1, with the half-inhibitory concentration (IC50) as the criterion. 50 Value representation.

[0081] The specific test subjects were: orlistat and dihydroquercetin (0.1:70), orlistat and dihydroquercetin (0.1:60), and orlistat and dihydroquercetin (0.2:60). All ratios are mass ratios.

[0082] The concentration gradients for each test subject are set as follows:

[0083] Orlistat and dihydroquercetin (0.1:70): (0.1+70)μg / mL, (0.05+35)μg / mL, (0.025+17.5)μg / mL, (0.0125+8.75)μg / mL;

[0084] Orlistat and dihydroquercetin (0.1:60): (0.1+60)μg / mL, (0.05+30)μg / mL, (0.025+15)μg / mL, (0.0125+7.5)μg / mL;

[0085] Orlistat and dihydroquercetin (0.2:60): (0.2+60)μg / mL, (0.1+30)μg / mL, (0.05+15)μg / mL, (0.025+7.5)μg / mL.

[0086] Concentration gradient setting principle: Taking orlistat and dihydroquercetin (0.1:60) as an example, the initial concentration of the composition is set to 60.1 μg / mL, that is, the initial concentration of orlistat in the composition sample solution is 0.1 μg / mL, and the initial concentration of dihydroquercetin is 60 μg / mL. Based on the initial concentration of 60.1 μg / mL, the composition sample solution is successively diluted 2-fold to 60.1 μg / mL, 30.05 μg / mL, 15.025 μg / mL, and 7.5125 μg / mL. Similarly, in the orlistat and dihydroquercetin (0.1:70) composition, the initial concentration is set to 70.1 μg / mL, and it is successively diluted 2-fold to form a concentration gradient. Other compositions also follow this principle.

[0087] The experimental results are shown in Table 4.

[0088] Table 4

[0089]

[0090] The data are derived from the results of three independent experiments and are expressed as "mean ± standard deviation".

[0091] Tables 1 and 4 show that the combined use of orlistat and dihydroquercetin, while achieving similar inhibition rates, significantly reduces the dosage of each component compared to their individual use, and enhances their inhibitory activity against pancreatic lipase. The effect is particularly pronounced when the mass ratio of the two is 0.1:60.

[0092] Example 5

[0093] Using a combination of orlistat and 3-O-methylquercetin as the test subject, the inhibitory activity of each test subject against pancreatic lipase was tested using the method described in Example 1, with the half-inhibitory concentration (IC50) as the criterion. 50 Value representation.

[0094] The specific test subjects were: orlistat and 3-O-methylquercetin (0.1:60), orlistat and 3-O-methylquercetin (0.1:50), and orlistat and 3-O-methylquercetin (0.2:50). All ratios mentioned above are mass ratios.

[0095] The concentration gradients for each test subject are set as follows:

[0096] Orlistat and 3-O-methylquercetin (0.1:60): (0.1+60) μg / mL, (0.05+30) μg / mL, (0.025+15) μg / mL, (0.0125+7.5) μg / mL;

[0097] Orlistat and 3-O-methylquercetin (0.1:50): (0.1+50) μg / mL, (0.05+25) μg / mL, (0.025+12.5) μg / mL, (0.0125+6.25) μg / mL;

[0098] Orlistat and 3-O-methylquercetin (0.2:50): (0.2+50)μg / mL, (0.1+25)μg / mL, (0.05+12.5)μg / mL, (0.025+6.25)μg / mL.

[0099] Concentration gradient setting principle: Taking orlistat and 3-O-methylquercetin (0.1:50) as an example, the initial concentration of the composition is set to 50.1 μg / mL, that is, the initial concentration of orlistat in the composition sample solution is 0.1 μg / mL, and the initial concentration of 3-O-methylquercetin is 50 μg / mL. Based on the initial concentration of 50.1 μg / mL, the composition sample solution is successively diluted 2-fold to 50.1 μg / mL, 25.05 μg / mL, 12.525 μg / mL, and 6.2625 μg / mL. Similarly, in the orlistat and 3-O-methylquercetin (0.1:60) composition, the initial concentration is set to 60.1 μg / mL, and it is successively diluted 2-fold to form a concentration gradient. Other compositions also follow this principle.

[0100] The experimental results are shown in Table 5.

[0101] Table 5

[0102]

[0103] The data are derived from the results of three independent experiments and are expressed as "mean ± standard deviation".

[0104] As shown in Tables 1 and 5, the combined use of orlistat and 3-O-methylquercetin, while achieving similar inhibition rates, significantly reduces the dosage of each component compared to its individual use, and enhances the inhibitory activity against pancreatic lipase. The effect is particularly pronounced when the mass ratio of the two is 0.1:50.

[0105] Example 6

[0106] Using orlistat and EGCG as test subjects, the inhibitory activity of each test subject against pancreatic lipase was tested using the method described in Example 1, with the half-inhibitory concentration (IC50) as the criterion. 50 Value representation.

[0107] The specific test subjects were: orlistat and EGCG (0.1:30), orlistat and EGCG (0.1:20), and orlistat and EGCG (0.2:20). All ratios mentioned above are mass ratios.

[0108] The concentration gradients for each test subject are set as follows:

[0109] Orlistat and EGCG (0.1:30): (0.1+30) μg / mL, (0.05+15) μg / mL, (0.025+7.5) μg / mL, (0.0125+3.75) μg / mL;

[0110] Orlistat and EGCG (0.1:20): (0.1+20) μg / mL, (0.05+10) μg / mL, (0.025+5) μg / mL, (0.0125+2.5) μg / mL;

[0111] Orlistat and EGCG (0.2:20): (0.2+20)μg / mL, (0.1+10)μg / mL, (0.05+5)μg / mL, (0.025+2.5)μg / mL.

[0112] Concentration gradient setting principle: Taking orlistat and EGCG (0.1:20) as an example, the initial concentration of the composition is set to 20.1 μg / mL, that is, the initial concentration of orlistat in the sample solution is 0.1 μg / mL, and the initial concentration of EGCG is 20 μg / mL. Based on the initial concentration of 20.1 μg / mL, the sample solution is successively diluted 2-fold to 20.1 μg / mL, 10.05 μg / mL, 5.025 μg / mL, and 2.5125 μg / mL. Similarly, in the orlistat and EGCG (0.1:30) composition, the initial concentration is set to 30.1 μg / mL, and it is successively diluted 2-fold to form a concentration gradient. Other compositions also follow this principle.

[0113] The experimental results are shown in Table 6.

[0114] Table 6

[0115]

[0116] The data are derived from the results of three independent experiments and are expressed as "mean ± standard deviation".

[0117] As shown in Tables 1 and 6, the combined use of orlistat and EGCG, while achieving similar inhibition rates, significantly reduces the dosage of each component compared to their individual use, and enhances their inhibitory activity against pancreatic lipase. The effect is particularly pronounced when the mass ratio of the two is 0.1:20.

[0118] Determination of the combination drug coefficient

[0119] The combination drug coefficient (CI) of the compositions in Examples 2, 3, 4, 5 and 6 was determined, the CI values ​​were calculated using the CompuSyn software, and the synergistic effect between the drugs was evaluated.

[0120] The combined drug efficacy coefficient of a 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 combined drug efficacy coefficient of a composition, it is necessary to set a concentration gradient for each monomer compound in the composition to obtain the corresponding inhibition rate.

[0121] Taking orlistat and luteolin (0.1:8) in Example 2 as an example, the initial concentration of the composition was set to 8.1 μg / mL, the initial concentration of orlistat was 0.1 μg / mL, and the initial concentration of luteolin was 8 μg / mL. Therefore, the gradient concentration setting of orlistat was based on the initial concentration of 0.1 μg / mL, and then successively diluted by 2 times to 0.1 μg / mL, 0.05 μg / mL, 0.025 μg / mL, and 0.0125 μg / mL. The gradient concentration settings for luteolin were based on an initial concentration of 8 μg / mL, with subsequent 2-fold dilutions to 8 μg / mL, 4 μg / mL, 2 μg / mL, and 1 μg / mL. The gradient concentration settings for the orlistat and luteolin (0.1:8) combination were based on an initial concentration of 8.1 μg / mL, with subsequent 2-fold dilutions to 8.1 μg / mL, 4.05 μg / mL, 2.025 μg / mL, and 1.0125 μg / mL.

[0122] The inhibition rates of orlistat, luteolin, orlistat and luteolin (0.1:8) at different concentration gradients were statistically analyzed, and the results are as follows:

[0123] The inhibition rates of orlistat at various concentration gradients were 8.3±0.1%, 15.4±2.1%, 22.4±1.6%, and 41.6±2.4% (from low to high concentration), respectively. The inhibition rates of luteolin at various concentration gradients were 11.9±1.5%, 22.5±1.3%, 36.5±1.5%, and 50.2±2.4% (from low to high concentration), respectively. The inhibition rates of the orlistat and luteolin (0.1:8) combination at various concentration gradients were 28.4±1.2%, 40.3±1.1%, 62.4±2.1%, and 72.5±3.2% (from low to high concentration), respectively.

[0124] The data were processed using CompuSyn software to obtain the CI values ​​of orlistat and luteolin (0.1:8) in the orlistat and luteolin (0.1:8) composition, as shown in Table 7. This indicates that the orlistat and luteolin (0.1:8) composition inhibits the Fa-CI trend of pancreatic lipase, as shown in Table 7. Figure 1 As shown.

[0125] Taking orlistat and quercetin (0.1:2) in Example 3 as an example, the initial concentration of the composition was set to 2.1 μg / mL, the initial concentration of orlistat was 0.1 μg / mL, and the initial concentration of quercetin was 2 μg / mL. Therefore, the gradient concentration setting of orlistat was based on the initial concentration of 0.1 μg / mL, and then successively diluted by 2-fold to 0.1 μg / mL, 0.05 μg / mL, 0.025 μg / mL, and 0.0125 μg / mL; quercetin... The gradient concentration settings were based on an initial concentration of 2 μg / mL, with subsequent 2-fold dilutions to 2 μg / mL, 1 μg / mL, 0.5 μg / mL, and 0.25 μg / mL. The gradient concentration settings for the orlistat and quercetin (0.1:2) combination were based on an initial concentration of 2.1 μg / mL, with subsequent 2-fold dilutions to 2.1 μg / mL, 1.05 μg / mL, 0.525 μg / mL, and 0.2625 μg / mL.

[0126] The inhibition rates of orlistat, quercetin, orlistat and quercetin (0.1:2) at different concentration gradients were statistically analyzed, and the results are as follows:

[0127] The inhibition rates of orlistat under the concentration gradient were 8.3±0.1%, 15.4±2.1%, 22.4±1.6%, and 41.6±2.4% (from low to high concentration), respectively; the inhibition rates of quercetin under the concentration gradient were 7.1±0.3%, 15.5±1.1%, 24.2±1.2%, and 43.1±1.3% (from low to high concentration), respectively; and the inhibition rates of the orlistat and quercetin (0.1:2) combination under the concentration gradient were 15.4±1.2%, 33.3±1.4%, 50.4±1.1%, and 67.5±2.2% (from low to high concentration), respectively.

[0128] The data were processed using CompuSyn software to obtain the CI values ​​of orlistat and quercetin in the orlistat and quercetin (0.1:2) composition, as shown in Table 7. This demonstrates the Fa-CI trend of the orlistat and quercetin (0.1:2) composition in inhibiting pancreatic lipase, as shown in Table 7. Figure 2 As shown.

[0129] Taking orlistat and dihydroquercetin (0.1:60) in Example 4 as an example, the initial concentration of the composition was set to 60.1 μg / mL, the initial concentration of orlistat was 0.1 μg / mL, and the initial concentration of dihydroquercetin was 60 μg / mL. Therefore, the gradient concentration setting of orlistat was based on the initial concentration of 0.1 μg / mL, and then successively diluted by 2-fold to 0.1 μg / mL, 0.05 μg / mL, 0.025 μg / mL, and 0.0125 μg / mL; the dihydroquercetin... The gradient concentration settings for orlistat and dihydroquercetin (0.1:60) were based on an initial concentration of 60 μg / mL, with subsequent 2-fold dilutions to 60 μg / mL, 30 μg / mL, 15 μg / mL, and 7.5 μg / mL. The gradient concentration settings for the orlistat and dihydroquercetin (0.1:60) combination were based on an initial concentration of 60.1 μg / mL, with subsequent 2-fold dilutions to 60.1 μg / mL, 30.05 μg / mL, 15.025 μg / mL, and 7.5125 μg / mL.

[0130] The inhibition rates of orlistat, dihydroquercetin, orlistat and dihydroquercetin (0.1:60) at different concentration gradients were statistically analyzed, and the results are as follows:

[0131] The inhibition rates of orlistat under the concentration gradient were 8.3±0.1%, 15.4±2.1%, 22.4±1.6%, and 41.6±2.4% (from low to high concentration), respectively. The inhibition rates of dihydroquercetin under the concentration gradient were 5.5±0.2%, 19.5±1.1%, 27.2±1.3%, and 47.2±1.1% (from low to high concentration), respectively. The inhibition rates of the orlistat and dihydroquercetin (0.1:60) combination under the concentration gradient were 23.4±1.2%, 40.3±1.3%, 60.4±2.2%, and 75.5±1.2% (from low to high concentration), respectively.

[0132] The data were processed using CompuSyn software to obtain the CI values ​​of orlistat and dihydroquercetin in the orlistat and dihydroquercetin (0.1:60) composition, as shown in Table 7. This indicates the Fa-CI trend of the orlistat and dihydroquercetin (0.1:60) composition in inhibiting pancreatic lipase, as shown in Table 7. Figure 3 As shown.

[0133] Taking orlistat and 3-O-methylquercetin (0.1:50) in Example 5 as an example, the initial concentration of the composition was set to 50.1 μg / mL, the initial concentration of orlistat was 0.1 μg / mL, and the initial concentration of 3-O-methylquercetin was 50 μg / mL. Therefore, the gradient concentration setting of orlistat was based on the initial concentration of 0.1 μg / mL, and then successively diluted by 2-fold to 0.1 μg / mL, 0.05 μg / mL, 0.025 μg / mL, and 0.0125 μg / mL; 3-O- The gradient concentration settings for methyl quercetin were based on an initial concentration of 50 μg / mL, followed by sequential 2-fold dilutions to 50 μg / mL, 25 μg / mL, 12.5 μg / mL, and 6.25 μg / mL. The gradient concentration settings for the combination of orlistat and 3-O-methyl quercetin (0.1:50) were based on an initial concentration of 50.1 μg / mL, followed by sequential 2-fold dilutions to 50.1 μg / mL, 25.05 μg / mL, 12.525 μg / mL, and 6.2625 μg / mL.

[0134] The inhibition rates of orlistat, 3-O-methylquercetin, orlistat and 3-O-methylquercetin (0.1:50) at different concentration gradients were statistically analyzed, and the results are as follows:

[0135] The inhibition rates of orlistat under the concentration gradient were 8.3±0.1%, 15.4±2.1%, 22.4±1.6%, and 41.6±2.4% (from low to high concentration), respectively. The inhibition rates of 3-O-methylquercetin under the concentration gradient were 6.1±0.1%, 15.7±1.1%, 23.2±1.3%, and 45.1±1.4% (from low to high concentration), respectively. The inhibition rates of orlistat and 3-O-methylquercetin (0.1:50) under the concentration gradient were 24.4±1.2%, 41.3±1.3%, 60.4±2.1%, and 75.5±3.2% (from low to high concentration), respectively.

[0136] The data were processed using CompuSyn software to obtain the CI values ​​of orlistat and 3-O-methylquercetin in the orlistat and 3-O-methylquercetin (0.1:50) composition, as shown in Table 7. This indicates that the orlistat and 3-O-methylquercetin (0.1:50) composition inhibits pancreatic lipase via the Fa-CI method. Figure 4 As shown.

[0137] Taking orlistat and EGCG (0.1:20) in Example 6 as an example, the initial concentration of the composition was set to 20.1 μg / mL, the initial concentration of orlistat was 0.1 μg / mL, and the initial concentration of EGCG was 20 μg / mL. Therefore, the gradient concentration setting of orlistat was based on the initial concentration of 0.1 μg / mL, and then successively diluted by 2-fold to 0.1 μg / mL, 0.05 μg / mL, 0.025 μg / mL, and 0.0125 μg / mL; EGCG... The gradient concentration settings for G were based on an initial concentration of 20 μg / mL, with subsequent 2-fold dilutions to 20 μg / mL, 10 μg / mL, 5 μg / mL, and 2.5 μg / mL. The gradient concentration settings for the orlistat and EGCG (0.1:20) combination were based on an initial concentration of 20.1 μg / mL, with subsequent 2-fold dilutions to 20.1 μg / mL, 10.05 μg / mL, 5.025 μg / mL, and 2.5125 μg / mL.

[0138] The inhibition rates of orlistat, EGCG, orlistat and EGCG (0.1:20) at different concentration gradients were statistically analyzed, and the results are as follows:

[0139] The inhibition rates of orlistat under the concentration gradient were 8.3±0.1%, 15.4±2.1%, 22.4±1.6%, and 41.6±2.4% (from low to high concentration), respectively. The inhibition rates of EGCG under the concentration gradient were 9.5±1.3%, 15.5±1.2%, 28.5±1.1%, and 45.2±2.2% (from low to high concentration), respectively. The inhibition rates of the orlistat and EGCG (0.1:20) combination under the concentration gradient were 23.4±1.2%, 42.3±1.4%, 56.4±1.2%, and 75.5±2.5% (from low to high concentration), respectively.

[0140] The data were processed using CompuSyn software to obtain the CI values ​​of orlistat and EGCG (0.1:20) in the orlistat and EGCG (0.1:20) combination, as shown in Table 7. This indicates the Fa-CI trend of the orlistat and EGCG (0.1:20) combination in inhibiting pancreatic lipase, as shown in Table 7. Figure 5 As shown.

[0141] Similarly, for other compositions, including orlistat and luteolin (0.1:10), orlistat and luteolin (0.2:8), orlistat and quercetin (0.1:4), orlistat and quercetin (0.2:2), orlistat and dihydroquercetin (0.1:70), orlistat and dihydroquercetin (0.2:60), orlistat and 3-O-methylquercetin (0.1:60), orlistat and 3-O-methylquercetin (0.2:50), orlistat and EGCG (0.1:30), and orlistat and EGCG (0.2:20), the concentration gradient settings of each monomer compound and the calculation of CI values ​​shall all be based on the above principles.

[0142] The measurement results are shown in Table 7:

[0143] Table 7

[0144]

[0145]

[0146] The data are derived from the results of three independent experiments and are expressed as "mean ± standard deviation".

[0147] As shown in Table 7:

[0148] The combined use of orlistat and luteolin at a mass ratio of 0.1:10 showed a synergistic effect with a combination drug coefficient (CI) less than 1. The mean CI of the combination drug index was [value missing]. avg The value is 0.82. When the mass ratio of the two is 0.1:8, GI... 50 GI 75 and GI 90 All values ​​were less than 0.60, indicating a strong synergistic effect when the mass ratio was 0.1:8. The mean combination drug index (CI) was also less than 0.60. avg The value was 0.54. When the mass ratio of the two drugs was 0.2:8, the combination drug coefficient (CI) was less than 1, indicating a synergistic effect. The mean combination drug index (CI) was... avg The value is 0.82.

[0149] The combined use of orlistat and quercetin at a mass ratio of 0.1:4 resulted in a combination drug coefficient (CI) of approximately 1, demonstrating an additive effect. The mean CI was [value missing]. avg The value is 1.10. When the mass ratio of the two is 0.1:2, GI... 50 GI 75 and GI 90 All values ​​were less than 0.70, indicating a strong synergistic effect between the two drugs at a mass ratio of 0.1:2. The mean combination drug index (CI) was also less than 0.70. avgThe value was 0.66. When the mass ratio of the two drugs was 0.2:2, the combination drug coefficient (CI) was approximately 1, exhibiting an additive effect. The mean combination drug index (CI) was... avg The value is 0.99.

[0150] The combined use of orlistat and dihydroquercetin at a mass ratio of 0.1:70 showed a synergistic effect with a combination drug index (CI) less than 1. The mean CI was [value missing]. avg The value is 0.57. When the mass ratio of the two is 0.1:60, GI... 50 GI 75 and GI 90 All values ​​were less than 0.60, indicating a strong synergistic effect when the mass ratio was 0.1:60. The mean combination drug index (CI) was also less than 0.60. avg The value was 0.43. When the mass ratio of the two drugs was 0.2:60, the combination drug coefficient (CI) was less than 1, indicating a synergistic effect. The mean combination drug index (CI) was... avg The value is 0.68.

[0151] The combined use of orlistat and 3-O-methylquercetin at a mass ratio of 0.1:60 showed a synergistic effect with a combination drug index (CI) less than 1. The mean CI was [value missing]. avg The value is 0.60. When the mass ratio of the two is 0.1:50, GI... 50 GI 75 and GI 90 All values ​​were less than 0.50, indicating a strong synergistic effect when the mass ratio was 0.1:50. The mean combination drug index (CI) was also less than 0.50. avg The value was 0.48. When the mass ratio of the two drugs was 0.2:50, the combination drug coefficient (CI) was less than 1, indicating a synergistic effect. The mean combination drug index (CI) was... avg The value is 0.68.

[0152] The combined use of orlistat and EGCG at a mass ratio of 0.1:30 showed a synergistic effect with a combination drug index (CI) less than 1. The mean CI was [value missing]. avg The value is 0.60. When the mass ratio of the two is 0.1:20, GI... 50 GI 75 and GI 90 All values ​​were less than 0.60, indicating a strong synergistic effect between the two drugs at a mass ratio of 0.1:20. The mean combination drug index (CI) was also less than 0.60. avg The value was 0.51. When the mass ratio of the two drugs was 0.2:20, the combination drug coefficient (CI) was less than 1, indicating a weak synergistic effect. The mean combination drug index (CI) was...avg The value is 0.83.

[0153] Comparative Example

[0154] In addition to the embodiments described above, this invention also provides other cases where orlistat cannot form a synergistic effect during the combination screening process, in which compound A involves apigenin, morin, dihydromorin, and rutin. These cases represent only a portion of the work done at the time of inventing this invention. See below:

[0155] First, the IC50 of each monomeric compound against pancreatic lipase was tested according to the method described in the above embodiments. 50 The values ​​are shown in Table 8 below:

[0156] Table 8

[0157]

[0158] Then, take IC values ​​close to those of each monomer compound. 50 The values ​​were used to determine the pancreatic lipase inhibition rate, as shown in Table 9:

[0159] Table 9

[0160] Celery 20 45.1±1.5％ Morin 4 52.7±3.1％ Dihydromosin 30 52.8±1.8％ Yellow pigment 6 53.5±3.2％

[0161] The above-mentioned monomeric compounds were combined with orlistat, and the pancreatic lipase inhibition rate of the combinations was determined, as shown in Table 10 below:

[0162] Table 10

[0163]

[0164]

[0165] As shown in Table 10, the combined inhibition rate of each of the above monomeric compounds with orlistat is directly lower than that of the individual compounds. The combination actually exhibits an antagonistic effect and has no synergistic effect.

[0166] Furthermore, this invention also tested the pancreatic lipase inhibition rates of luteolin with quercetin, dihydroquercetin, 3-O-methylquercetin, and EGCG at mass ratios of 8:2, 8:60, 8:50, and 8:20, respectively. The results showed that the inhibition rates of the compositions were 41.3±1.1%, 44.8±2.2%, 43.1±2.1%, and 41.3±2.4%, respectively, all lower than that of luteolin. The inhibition rates of luteolin, quercetin, dihydroquercetin, 3-O-methylquercetin, and EGCG on pancreatic lipase were 50.2±2.4%, 43.1±1.3%, 47.2±1.1%, 45.1±1.4%, and 45.2±2.2%, respectively. This indicates that luteolin also has antagonistic effects with quercetin, dihydroquercetin, 3-O-methylquercetin, and EGCG. This invention also tested the pancreatic lipase inhibition rates of quercetin and dihydroquercetin, 3-O-methylquercetin, and EGCG at mass concentration ratios of 2:60, 2:50, and 2:20, respectively. The results showed that the inhibition rates of the compositions were 41.5±2.4%, 44.1±1.1%, and 40.8±2.3%, respectively, all lower than the inhibition rates of quercetin, dihydroquercetin, 3-O-methylquercetin, and EGCG alone on pancreatic lipase (43.1±1.3%, 47.2±1.1%, 45.1±1.4%, and 45.2±2.2%, respectively). This indicates that there is actually an antagonistic effect between quercetin and dihydroquercetin, 3-O-methylquercetin, and EGCG. The present invention also tested the pancreatic lipase inhibition rate of dihydroquercetin, 3-O-methylquercetin, and EGCG at mass concentration ratios of 60:50 and 60:20, respectively. The results showed that the inhibition rates of the compositions were 41.6±1.5% and 42.5±1.3%, respectively, both lower than the inhibition rates of dihydroquercetin, 3-O-methylquercetin, and EGCG alone on pancreatic lipase (47.2±1.1%, 45.1±1.4%, and 45.2±2.2%, respectively). This indicates that there is actually an antagonistic effect between dihydroquercetin and 3-O-methylquercetin and EGCG. The present invention also tested the pancreatic lipase inhibition rate of 3-O-methylquercetin and EGCG at a mass concentration ratio of 50:20. The results showed that the inhibition rate of the composition was 39.8±1.3%, which was lower than the inhibition rate of 3-O-methylquercetin and EGCG alone on pancreatic lipase (45.1±1.4% and 45.2±2.2%, respectively). This indicates that there is actually an antagonistic effect between 3-O-methylquercetin and EGCG.

[0167] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention in any other way. Any person skilled in the art may make changes or modifications to the above-disclosed technical content to create equivalent embodiments. However, any simple modifications, equivalent changes, and modifications made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the protection scope of the present invention.

Claims

1. An orlistat composition for the treatment of obesity, characterized in that, Consist of orlistat and luteolin; wherein the mass ratio of orlistat and luteolin is 0.1:10-0.2:

8.

2. The orlistat composition for use in the treatment of obesity according to claim 1, wherein The mass ratio of orlistat and luteolin is 0.1:

8.

3. Use of the orlistat composition for treating obesity according to claim 1 or 2 in the preparation of a medicament for treating obesity.

4. A medicament for the treatment of obesity, characterized in that, The medicament contains the orlistat composition according to claim 1 or 2.

5. The medicament according to claim 4, characterized in that, Further comprising a pharmaceutically acceptable carrier.

6. The medicament according to claim 4, characterized in that, The dosage form of the medicament is selected from powder, granule, capsule, injection, oral liquid or tablet.

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