CN116270608A

CN116270608A - Orlistat combination composition and application thereof

Info

Publication number: CN116270608A
Application number: CN202310242572.XA
Authority: CN
Inventors: 张强; 焦中高; 潘俊坤; 陈大磊; 刘杰超; 陈磊山; 田金河; 刘慧�; 杨文博; 吕真真
Original assignee: Xinxiang University; Zhengzhou Fruit Research Institute CAAS
Current assignee: Xinxiang University; Zhengzhou Fruit Research Institute CAAS
Priority date: 2023-03-14
Filing date: 2023-03-14
Publication date: 2023-06-23

Abstract

The invention discloses an orlistat combination composition and application thereof, and belongs to the technical field of natural medicines. The composition of the invention consists of orlistat and 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. There is a synergistic effect between the ingredients in the composition. The combined medication of the components can greatly reduce the dosage of the components when the components are independently used, reduce the toxic and side effects of the medicine, and can obtain the synergistic treatment effect. Therefore, when the corresponding effect of treating obesity is achieved, the combined drug can effectively reduce the occurrence of drug resistance of organisms, and has better medical application prospect.

Description

Orlistat combination composition and application thereof

Technical Field

The invention belongs to the technical field of natural medicines, and particularly relates to an orlistat combination composition and application thereof.

Background

In recent years, with changes in modern lifestyle and dietary structure, the incidence and mortality of obesity and obesity-related metabolic diseases have risen year by year, becoming an increasingly serious global public health problem. Although genetic, physiological, medical and behavioral factors are all causative factors of obesity, the most major cause of obesity is a long-term imbalance between energy intake and energy expenditure. Currently, a variety of strategies are used to treat overweight and obesity, including regulation of lipid metabolism, inhibition of nutrient absorption, regulation of fat signaling, regulation of satiety, etc., and 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 into 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 received much attention as an important anti-obesity target. To date, orlistat is a weight loss drug marketed as an irreversible pancreatic lipase inhibitor that can directly target serine residues in the mammalian pancreatic lipase catalytic triad by forming covalent bonds. While orlistat has excellent weight loss efficacy, various serious side effects of the drug (including urgent intestinal tract, frequent defecation, oil drainage, diarrhea, and liver and kidney damage, etc.) severely hamper its long-term use in clinic. Therefore, the combination of the medicine and orlistat can inhibit the activity of pancreatic lipase, produce synergistic effect and relatively reduce the dosage, and has important significance for improving human obesity.

Disclosure of Invention

The invention provides an orlistat composition, which consists of orlistat and a compound A; wherein, the compound A is selected from luteolin, quercetin, dihydroquercetin, 3-O-methyl quercetin or EGCG;

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

The orlistat composition has an inhibitory effect on pancreatic lipase, and based on the orlistat composition, the invention provides application of the orlistat composition in preparation of medicines with the pancreatic lipase inhibitory effect.

Those skilled in the art know that pancreatic lipase is closely related to obesity, and based on this, the present invention provides the use of the above orlistat composition in the preparation of a medicament for treating obesity.

In the orlistat composition, when the mass ratio of the orlistat to the luteolin is 0.1:10-0.2:8, a synergistic effect exists between the orlistat and the luteolin in the aspect of pancreatic lipase inhibition; when the mass ratio of orlistat to quercetin is 0.1:2-0.2:2, a synergistic effect exists between the orlistat and the quercetin in the aspect of pancreatic lipase inhibition; when the mass ratio of the orlistat to the dihydroquercetin is 0.1:70-0.2:60, a synergistic effect exists between the orlistat and the dihydroquercetin in the aspect of pancreatic lipase inhibition; when the mass ratio of orlistat to 3-O-methyl quercetin is 0.1:60-0.2:50, a synergistic effect exists between the two in the aspect of pancreatic lipase inhibition; when the mass ratio of orlistat to EGCG is 0.1:30-0.2:20, there is a synergistic effect in pancreatic lipase inhibition. Based on the above, in a specific embodiment, the orlistat composition provided by the invention consists of orlistat and luteolin according to a mass ratio of 0.1:10-0.2:8; or the orlistat/quercetin composite material consists of orlistat and quercetin in the mass ratio of 0.1 to 2-0.2 to 2; or the orlistat and the dihydroquercetin according to the mass ratio of 0.1:70-0.2:60; or the orlistat and the 3-O-methyl quercetin with the mass ratio of 0.1:60-0.2:50; or the mixture is composed of orlistat and EGCG according to the mass ratio of 0.1:30-0.2:20.

In a specific embodiment, the orlistat composition provided by the invention consists of orlistat and luteolin according to a mass ratio of 0.1:8; or the preparation comprises orlistat and quercetin according to a mass ratio of 0.1:2; or the composition comprises orlistat and dihydroquercetin according to the mass ratio of 0.1:60; or the orlistat/3-O-methyl quercetin composition comprises orlistat and 3-O-methyl quercetin in a mass ratio of 0.1:50; or the composition is composed of orlistat and EGCG according to the mass ratio of 0.1:20.

According to the above, there can be correspondingly provided a medicament having a synergistic pancreatic lipase inhibitory effect, which comprises the above orlistat composition. The medicine contains pharmaceutically acceptable carriers, solvents, diluents, excipients or other mediums, and can be prepared into corresponding dosage forms such as powder, granules, capsules, injections, oral liquid, tablets and the like according to different requirements.

The beneficial effects of the invention are as follows:

the composition can inhibit the activity of pancreatic lipase, thereby achieving the effect of treating obesity. There is a synergistic effect between the ingredients in the composition. The combined medication of the components can greatly reduce the dosage of the components when the components are independently used, reduce the toxic and side effects of the medicine, and can obtain the synergistic treatment effect. Therefore, when the corresponding effect of treating obesity is achieved, the combined drug can effectively reduce the occurrence of drug resistance of organisms, and has better medical application prospect.

Drawings

FIG. 1 is a Fa-CI trend graph of a combination of orlistat and luteolin (0.1:8) versus pancreatic lipase;

FIG. 2 is a graph of Fa-CI trend of a combination of orlistat and quercetin (0.1:2) versus pancreatic lipase;

FIG. 3 is a Fa-CI trend graph of a combination of orlistat and dihydroquercetin (0.1:60) versus pancreatic lipase;

FIG. 4 is a graph of Fa-CI trend of a combination of orlistat and 3-O-methyl quercetin (0.1:50) versus pancreatic lipase;

FIG. 5 is a graph of Fa-CI trend of a combination of orlistat and EGCG (0.1:20) versus pancreatic lipase.

Detailed Description

Orlistat (Orlistat), formula C ₂₉ H ₅₃ NO ₅ The method comprises the steps of carrying out a first treatment on the surface of the Molecular weight: 495.7; CAS accession number: 96829-58-2, the structural formula is:

luteolin (Luteolin) with molecular formula of C ₁₅ H ₁₀ O ₆ The method comprises the steps of carrying out a first treatment on the surface of the Molecular weight: 286.24; CAS accession number: 491-70-3, structural formula:

quercetin (Quercetin) with molecular formula of C ₁₅ H ₁₀ O ₇ The method comprises the steps of carrying out a first treatment on the surface of the Molecular weight: 302.236; CAS accession number: 117-39-5, structural formula:

dihydroquercetin (Taxifolin) with molecular formula of C ₁₅ H ₁₂ O ₇ The method comprises the steps of carrying out a first treatment on the surface of the Molecular weight: 304.25;CAS accession number: 480-18-2, structural formula:

3-O-methyl quercetin (3-O-methyl quercetin) with molecular formula of C ₁₆ H ₁₂ O ₇ The method comprises the steps of carrying out a first treatment on the surface of the Molecular weight: 316.262; CAS accession number: 1486-70-0, structural formula:

EGCG with molecular formula of C ₂₂ H ₁₈ O ₁₁ The method comprises the steps of carrying out a first treatment on the surface of the Molecular weight: 458.38; CAS accession number: 989-51-5, the structural formula is:

other materials used in the present invention, such as those not specifically stated, are available through commercial sources. Other terms used herein, unless otherwise indicated, generally have meanings commonly understood by those of ordinary skill in the art. The invention will be described in further detail below in connection with specific embodiments and with reference to the data. The following examples are intended to illustrate the invention and are not intended to limit the scope of the invention in any way.

Example 1

An in vitro inhibition assay of pancreatic lipase activity was performed as follows:

dissolving a test object in dimethyl sulfoxide (DMSO) to prepare a mother solution of 10mg/mL, diluting the mother solution with PBS (after dilution, the DMSO content is less than 5%), and setting a concentration gradient to form a sample to be tested. A solution of pancreatic lipase (supernatant was collected by centrifugation) at 1mg/mL and a solution of substrate 4-Methylumbelliferyl oleate (4-MUO) at 0.1mM were prepared in PBS buffer.

To each well, 25. Mu.L of a sample to be measured and 25. Mu.L of pancreatic lipase (1 mg/mL) were added, and after mixing, 50. Mu.L of a reaction substrate 4-MUO was added, and incubated at 25℃for 20 minutes, and then 100. Mu.L of a sodium citrate buffer solution was added, and the fluorescence value of each well was measured with an microplate reader, the excitation wavelength was 320nm, and the emission wavelength was 450nm.

Pancreatic lipase activity inhibition rate at each gradient was calculated from FL values:

inhibition ratio = [1- (FL) _{Sample of} –FL _{Sample blank} )/(FL _{Negative control} -FL _{Blank space} )]×100％

Wherein FL is _{Sample of} Refer to the fluorescence value, FL, of a sample set _{Sample blank} Refer to the fluorescence value, FL, of the sample blank _{Negative control} Refer to the fluorescence value, FL, of the negative control group _{Blank space} Refers to the fluorescence values of the blank group.

From the pancreatic lipase activity inhibition ratios at each gradient, the half-inhibitory concentration (IC ₅₀ Values), the calculation and statistics process uses SPSS 20.0.

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

sample group: 25. Mu.L of the sample to be tested+25. Mu.L of enzyme;

sample blank: 25. Mu.L of sample to be tested+25. Mu.L of PBS;

negative control group: 25 μL PBS+25 μL enzyme;

blank group: 50. Mu.L PBS.

The experimental instrument and the reagent are as follows: pancreatic lipase (Type II, L3126), 4-Methylumbelliferyl oleate (4-MUO, S75164) and orlistat were purchased from Sigma company; luteolin, quercetin, dihydroquercetin, 3-O-methylquercetin and EGCG (Beijing solebao); millipore Simplicity Water purification System (Millipore, france), microplate reader TECAN infinite M200 PRO (Teacan Group Ltd., swizer).

Specifically, orlistat, luteolin, quercetin, dihydroquercetin, 3-O-methyl quercetin and EGCG were used as test subjects, and the inhibitory activity of each test subject on pancreatic lipase was tested by the above method and at half inhibitory concentration IC ₅₀ The value represents.

The concentration gradient of each test object was set as follows: orlistat: 0.2. Mu.g/mL, 0.1. Mu.g/mL, 0.05. Mu.g/mL, 0.025. Mu.g/mL; luteolin: 20. Mu.g/mL, 10. Mu.g/mL, 5. Mu.g/mL, 2.5. Mu.g/mL; quercetin: 4. Mu.g/mL, 2. Mu.g/mL, 1. Mu.g/mL, 0.5. Mu.g/mL; dihydroquercetin: 70 μg/mL, 35 μg/mL, 17.5 μg/mL, 8.75 μg/mL; 3-O-methyl quercetin: 60 μg/mL, 30 μg/mL, 15 μg/mL, 7.5 μg/mL; EGCG: 30. Mu.g/mL, 15. Mu.g/mL, 7.5. Mu.g/mL, 3.75. Mu.g/mL.

The test results are shown in table 1 below.

TABLE 1

Data were derived from the results of three independent experiments and are expressed as "mean ± standard deviation".

Example 2

The method of example 1 was used to test the pancreatic lipase inhibition activity of each subject using a combination of orlistat and luteolin as the subject, at a half inhibition concentration IC ₅₀ The value represents.

Each test object is specifically: orlistat and luteolin (0.1:10), orlistat and luteolin (0.1:8), orlistat and luteolin (0.2:8). The above proportions are mass ratios.

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

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;

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;

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.

Concentration gradient setting principle: taking orlistat and luteolin (0.1:8) as examples, the initial concentration of the composition was set to 8.1 μg/mL, i.e., the initial concentration of orlistat in the composition sample solution was 0.1 μg/mL and the initial concentration of luteolin was 8 μg/mL. The composition sample solutions were sequentially diluted 2-fold at an initial concentration of 8.1. Mu.g/mL, followed by 8.1. Mu.g/mL, 4.05. Mu.g/mL, 2.025. Mu.g/mL, and 1.0125. Mu.g/mL. Similarly, in a composition of orlistat and luteolin (0.1:10), the initial concentration of the composition was set to 10.1 μg/mL, and the composition was diluted 2-fold in sequence to form a concentration gradient. Other compositions follow this principle as well.

The test results are shown in Table 2.

TABLE 2

As can be seen from tables 1 and 2: the combined use of the orlistat and the luteolin can obviously reduce the dosage of each component when the components are independently used under the condition of achieving similar inhibition rate, and improve the inhibition activity on pancreatic lipase. Especially when the mass ratio of the two is 0.1:8, the effect is more obvious.

Example 3

Using the method of example 1, the inhibitory activity of each test subject against pancreatic lipase was tested using a combination of orlistat and quercetin as the test subject, at a half inhibitory concentration IC ₅₀ The value represents.

Each test object is specifically: orlistat and quercetin (0.1:4), orlistat and quercetin (0.1:2), orlistat and quercetin (0.2:2). The above proportions are mass ratios.

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

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;

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;

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.

Concentration gradient setting principle: taking orlistat and quercetin (0.1:2) as examples, the initial concentration of the composition was set to 2.1 μg/mL, i.e., the initial concentration of orlistat in the composition sample solution was 0.1 μg/mL, and the initial concentration of quercetin was 2 μg/mL. The composition sample solutions were sequentially diluted 2-fold based on the initial concentration of 2.1. Mu.g/mL, with 2.1. Mu.g/mL, 1.05. Mu.g/mL, 0.525. Mu.g/mL, and 0.2625. Mu.g/mL. Similarly, in the composition of orlistat and quercetin (0.1:4), the initial concentration of the composition was set to 4.1 μg/mL, and the composition was diluted 2-fold in sequence to form a concentration gradient. Other compositions follow this principle as well.

The test results are shown in Table 3.

TABLE 3 Table 3

As can be seen from tables 1 and 3: under the condition of achieving similar inhibition rate, the combined use of orlistat and quercetin can obviously reduce the dosage of each component when the components are independently used, and improve the inhibition activity on pancreatic lipase. Especially when the mass ratio of the two is 0.1:2, the effect is more obvious.

Example 4

The method of example 1 was used to test the pancreatic lipase inhibitory activity of each test subject using a combination of orlistat and dihydroquercetin as the test subjects, at a half inhibitory concentration IC ₅₀ Value tableShown.

Each test object is specifically: orlistat and dihydroquercetin (0.1:70), orlistat and dihydroquercetin (0.1:60), orlistat and dihydroquercetin (0.2:60). The above proportions are mass ratios.

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

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;

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;

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.

Concentration gradient setting principle: taking orlistat and dihydroquercetin (0.1:60) as examples, the initial concentration of the composition was set to 60.1 μg/mL, i.e., the initial concentration of orlistat in the composition sample solution was 0.1 μg/mL, and the initial concentration of dihydroquercetin was 60 μg/mL. The composition sample solutions were sequentially diluted 2-fold at an initial concentration of 60.1. Mu.g/mL, followed by 60.1. Mu.g/mL, 30.05. Mu.g/mL, 15.025. Mu.g/mL, 7.5125. Mu.g/mL. Similarly, in the composition of orlistat and dihydroquercetin (0.1:70), the initial concentration of the composition was set to 70.1 μg/mL, and the composition was diluted 2-fold in sequence to form a concentration gradient. Other compositions follow this principle as well.

The test results are shown in Table 4.

TABLE 4 Table 4

As can be seen from tables 1 and 4: the combined use of the orlistat and the dihydroquercetin can obviously reduce the dosage of each component when the components are independently used under the condition of achieving similar inhibition rate, and improve the inhibition activity on pancreatic lipase. Especially, when the mass ratio of the two is 0.1:60, the effect is more obvious.

Example 5

Using the method of example 1, the inhibitory activity of pancreatic lipase of each subject was tested using a combination of orlistat and 3-O-methyl quercetin as the subject, at a half inhibitory concentration IC ₅₀ The value represents.

Each test object is specifically: orlistat and 3-O-methyl quercetin (0.1:60), orlistat and 3-O-methyl quercetin (0.1:50), orlistat and 3-O-methyl quercetin (0.2:50). The above proportions are mass ratios.

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

orlistat and 3-O-methyl quercetin (0.1:60): (0.1+60) μg/mL, (0.05+30) μg/mL, (0.025+15) μg/mL, (0.0125+7.5) μg/mL;

orlistat and 3-O-methyl quercetin (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;

orlistat and 3-O-methyl quercetin (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.

Concentration gradient setting principle: taking orlistat and 3-O-methyl quercetin (0.1:50) as examples, the initial concentration of the composition was set to 50.1 μg/mL, i.e., the initial concentration of orlistat in the composition sample solution was 0.1 μg/mL, and the initial concentration of 3-O-methyl quercetin was 50 μg/mL. The composition sample solutions were sequentially diluted 2-fold at an initial concentration of 50.1. Mu.g/mL, followed by 50.1. Mu.g/mL, 25.05. Mu.g/mL, 12.525. Mu.g/mL, 6.2625. Mu.g/mL. Similarly, in a composition of orlistat and 3-O-methyl quercetin (0.1:60), the initial concentration of the composition was set to 60.1. Mu.g/mL, and the composition was diluted 2-fold in sequence to form a concentration gradient. Other compositions follow this principle as well.

The test results are shown in Table 5.

TABLE 5

As can be seen from tables 1 and 5: the combined use of the orlistat and the 3-O-methyl quercetin can obviously reduce the dosage of each component when being independently used under the condition of achieving similar inhibition rate, and improve the inhibition activity on pancreatic lipase. Especially, when the mass ratio of the two is 0.1:50, the effect is more obvious.

Example 6

The test subjects were tested for pancreatic lipase inhibitory activity in the semi-inhibitory concentration IC using the composition of orlistat and EGCG as the test subjects using the method of example 1 ₅₀ The value represents.

Each test object is specifically: orlistat and EGCG (0.1:30), orlistat and EGCG (0.1:20), orlistat and EGCG (0.2:20). The above proportions are mass ratios.

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

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;

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;

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.

Concentration gradient setting principle: taking orlistat and EGCG (0.1:20) as examples, the initial concentration of the composition was set to 20.1 μg/mL, i.e. the initial concentration of orlistat in the composition sample solution was 0.1 μg/mL and the initial concentration of EGCG was 20 μg/mL. The composition sample solutions were sequentially diluted 2-fold based on the initial concentration of 20.1. Mu.g/mL, and were sequentially 20.1. Mu.g/mL, 10.05. Mu.g/mL, 5.025. Mu.g/mL, and 2.5125. Mu.g/mL. Similarly, in a composition of orlistat and EGCG (0.1:30), the initial concentration of the composition was set to 30.1 μg/mL, and the composition was diluted 2-fold in sequence to form a concentration gradient. Other compositions follow this principle as well.

The test results are shown in Table 6.

TABLE 6

As can be seen from tables 1 and 6: the combined use of the orlistat and the EGCG can obviously reduce the dosage of each component when being singly used under the condition of achieving similar inhibition rate, and improve the inhibition activity on pancreatic lipase. Especially when the mass ratio of the two is 0.1:20, the effect is more obvious.

Determination of co-administration coefficient

The combinations of the compositions of examples 2, 3, 4, 5 and 6 were tested for their coefficient of co-administration (CI), and CI values were calculated according to the software CompuSyn and evaluated for synergy between the drugs.

The co-administration coefficient of the composition depends on the inhibition rate of the composition under different concentration gradients and the inhibition rate of each monomer compound in the composition under different concentration gradients, so that when the co-administration coefficient of the composition is measured, the concentration gradient setting needs to be carried out on each monomer compound in the composition to obtain the corresponding inhibition rate.

Taking orlistat and luteolin (0.1:8) as examples in example 2, 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; thus, the gradient concentration of orlistat was set by 2-fold dilution in order of 0.1. Mu.g/mL, 0.05. Mu.g/mL, 0.025. Mu.g/mL, 0.0125. Mu.g/mL based on the initial concentration of 0.1. Mu.g/mL; the gradient concentration of luteolin is set by sequentially carrying out 2-time dilution on the basis of the initial concentration of 8 mug/mL, and the concentration is sequentially 8 mug/mL, 4 mug/mL, 2 mug/mL and 1 mug/mL; the gradient concentration of the orlistat and luteolin (0.1:8) composition was set by sequentially performing 2-fold dilutions of 8.1 μg/mL, 4.05 μg/mL, 2.025 μg/mL, 1.0125 μg/mL on the basis of the initial concentration of 8.1 μg/mL.

The inhibition rates of orlistat, luteolin, orlistat and luteolin (0.1:8) at different concentration gradients were counted as follows:

the inhibition ratio of orlistat at each gradient concentration is 8.3+/-0.1%, 15.4+/-2.1%, 22.4+/-1.6%, 41.6+/-2.4% (from low concentration to high concentration), the inhibition ratio of luteolin at the concentration gradient is 11.9+/-1.5%, 22.5+/-1.3%, 36.5+/-1.5%, 50.2+/-2.4% (from low concentration to high concentration), and the inhibition ratio of the orlistat and luteolin (0.1:8) composition at the concentration gradient is 28.4+/-1.2%, 40.3+/-1.1%, 62.4+/-2.1%, 72.5+/-3.2% (from low concentration to high concentration).

The above data were processed using CompuSyn software to obtain CI values for orlistat and luteolin in the compositions of orlistat and luteolin (0.1:8), as shown in Table 7. Thus, the Fa-CI trend of the orlistat and luteolin (0.1:8) composition to inhibit pancreatic lipase can be derived as shown in FIG. 1.

Taking orlistat and quercetin (0.1:2) as examples in example 3, 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; thus, the gradient concentration of orlistat was set by 2-fold dilution in order of 0.1. Mu.g/mL, 0.05. Mu.g/mL, 0.025. Mu.g/mL, 0.0125. Mu.g/mL based on the initial concentration of 0.1. Mu.g/mL; the gradient concentration of quercetin is set by sequentially diluting 2 times based on the initial concentration of 2 mug/mL, and sequentially 2 mug/mL, 1 mug/mL, 0.5 mug/mL and 0.25 mug/mL; the concentration of the orlistat and quercetin (0.1:2) composition was set in a gradient by 2-fold dilution based on the initial concentration of 2.1. Mu.g/mL, followed by 2.1. Mu.g/mL, 1.05. Mu.g/mL, 0.525. Mu.g/mL, and 0.2625. Mu.g/mL.

The inhibition rates of orlistat, quercetin, orlistat and quercetin (0.1:2) at different concentration gradients were counted as follows:

the inhibition ratio of orlistat under the concentration gradient is 8.3+/-0.1%, 15.4+/-2.1%, 22.4+/-1.6%, 41.6+/-2.4% (from low concentration to high concentration), the inhibition ratio of quercetin under the concentration gradient is 7.1+/-0.3%, 15.5+/-1.1%, 24.2+/-1.2%, 43.1+/-1.3% (from low concentration to high concentration), and the inhibition ratio of the orlistat and quercetin (0.1:2) composition under the concentration gradient is 15.4+/-1.2%, 33.3+/-1.4%, 50.4+/-1.1%, 67.5+/-2.2% (from low concentration to high concentration).

The above data were processed using CompuSyn software to obtain CI values for orlistat and quercetin in the orlistat and quercetin (0.1:2) compositions, as shown in Table 7. Thus, the Fa-CI trend of the combination of orlistat and quercetin (0.1:2) to inhibit pancreatic lipase can be derived as shown in FIG. 2.

Taking orlistat and dihydroquercetin (0.1:60) as examples in example 4, 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; thus, the gradient concentration of orlistat was set by 2-fold dilution in order of 0.1. Mu.g/mL, 0.05. Mu.g/mL, 0.025. Mu.g/mL, 0.0125. Mu.g/mL based on the initial concentration of 0.1. Mu.g/mL; the gradient concentration of the dihydroquercetin is set by sequentially carrying out 2-time dilution on the basis of the initial concentration of 60 mug/mL, and the concentration is sequentially 60 mug/mL, 30 mug/mL, 15 mug/mL and 7.5 mug/mL; the gradient concentration of the orlistat and dihydroquercetin (0.1:60) composition was set by 2-fold dilution based on the initial concentration of 60.1 μg/mL, followed by 60.1 μg/mL, 30.05 μg/mL, 15.025 μg/mL, 7.5125 μg/mL.

The inhibition rates of orlistat, dihydroquercetin, orlistat and dihydroquercetin (0.1:60) at different concentration gradients were counted and the results were as follows:

the inhibition ratio of orlistat under the concentration gradient is 8.3+/-0.1%, 15.4+/-2.1%, 22.4+/-1.6%, 41.6+/-2.4% (from low concentration to high concentration), the inhibition ratio of dihydroquercetin under the concentration gradient is 5.5+/-0.2%, 19.5+/-1.1%, 27.2+/-1.3%, 47.2+/-1.1% (from low concentration to high concentration), and the inhibition ratio of orlistat and dihydroquercetin (0.1:60) composition under the gradient concentration is 23.4+/-1.2%, 40.3+/-1.3%, 60.4+/-2.2% and 75.5+/-1.2% (from low concentration to high concentration).

The above data were processed using CompuSyn software to obtain CI values for orlistat and dihydroquercetin in the orlistat and dihydroquercetin (0.1:60) compositions, as shown in Table 7. Thus, the Fa-CI trend of the combination of orlistat and dihydroquercetin (0.1:60) to inhibit pancreatic lipase can be derived as shown in FIG. 3.

Taking orlistat and 3-O-methyl quercetin (0.1:50) as examples in example 5, the initial concentration of the composition was set to 50.1. Mu.g/mL, the initial concentration of orlistat was 0.1. Mu.g/mL, and the initial concentration of 3-O-methyl quercetin was 50. Mu.g/mL; thus, the gradient concentration of orlistat was set by 2-fold dilution in order of 0.1. Mu.g/mL, 0.05. Mu.g/mL, 0.025. Mu.g/mL, 0.0125. Mu.g/mL based on the initial concentration of 0.1. Mu.g/mL; the gradient concentration of the 3-O-methyl quercetin is set by sequentially carrying out 2-time dilution on the basis of the initial concentration of 50 mug/mL, namely 50 mug/mL, 25 mug/mL, 12.5 mug/mL and 6.25 mug/mL; the gradient concentration of the orlistat and 3-O-methyl quercetin (0.1:50) composition was set by 2-fold dilution of the initial concentration of 50.1. Mu.g/mL, 25.05. Mu.g/mL, 12.525. Mu.g/mL, 6.2625. Mu.g/mL.

The inhibition rates of orlistat, 3-O-methyl quercetin, orlistat and 3-O-methyl quercetin (0.1:50) at different concentration gradients were counted and the results were as follows:

the inhibition ratio of orlistat under the concentration gradient is 8.3+/-0.1%, 15.4+/-2.1%, 22.4+/-1.6%, 41.6+/-2.4% (from low concentration to high concentration), the inhibition ratio of 3-O-methyl quercetin under the concentration gradient is 6.1+/-0.1%, 15.7+/-1.1%, 23.2+/-1.3%, 45.1+/-1.4% (from low concentration to high concentration), and the inhibition ratio of orlistat and 3-O-methyl quercetin (0.1:50) under the concentration gradient is 24.4+/-1.2%, 41.3+/-1.3%, 60.4+/-2.1%, 75.5+/-3.2% (from low concentration to high concentration).

The above data were processed using CompuSyn software to obtain CI values for orlistat and 3-O-methyl quercetin in an orlistat and 3-O-methyl quercetin (0.1:50) composition, as shown in Table 7. Thus, the Fa-CI trend of the combination of orlistat and 3-O-methyl quercetin (0.1:50) against pancreatic lipase can be derived as shown in FIG. 4.

Taking orlistat and EGCG (0.1:20) as examples in example 6, 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; thus, the gradient concentration of orlistat was set by 2-fold dilution in order of 0.1. Mu.g/mL, 0.05. Mu.g/mL, 0.025. Mu.g/mL, 0.0125. Mu.g/mL based on the initial concentration of 0.1. Mu.g/mL; the gradient concentration of EGCG is set by sequentially carrying out 2-time dilution on the basis of the initial concentration of 20 mug/mL, and the concentration is sequentially 20 mug/mL, 10 mug/mL, 5 mug/mL and 2.5 mug/mL; the concentration of the orlistat and EGCG (0.1:20) composition was set in a gradient manner by sequentially diluting the composition with 2 times based on the initial concentration of 20.1. Mu.g/mL, and the concentration was sequentially 20.1. Mu.g/mL, 10.05. Mu.g/mL, 5.025. Mu.g/mL and 2.5125. Mu.g/mL.

The inhibition rates of orlistat, EGCG, orlistat and EGCG (0.1:20) at different concentration gradients were counted as follows:

the inhibition ratio of orlistat under the concentration gradient is 8.3+/-0.1%, 15.4+/-2.1%, 22.4+/-1.6%, 41.6+/-2.4% (from low concentration to high concentration), the inhibition ratio of EGCG under the concentration gradient is 9.5+/-1.3%, 15.5+/-1.2%, 28.5+/-1.1%, 45.2+/-2.2% (from low concentration to high concentration), and the inhibition ratio of orlistat and EGCG (0.1:20) composition under the concentration gradient is 23.4+/-1.2%, 42.3+/-1.4%, 56.4+/-1.2% and 75.5+/-2.5% (from low concentration to high concentration).

The above data were processed using CompuSyn software to obtain CI values for orlistat and EGCG in an orlistat and EGCG (0.1:20) composition, as shown in Table 7. Thus, the Fa-CI trend of pancreatic lipase inhibition by the orlistat and EGCG (0.1:20) combination can be derived as shown in FIG. 5.

Similarly, 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-methyl quercetin (0.1:60), orlistat and 3-O-methyl quercetin (0.2:50), orlistat and EGCG (0.1:30), orlistat and EGCG (0.2:20), concentration gradient settings of individual compounds, calculation of CI values, and the like are all based on the above principles.

The measurement results are shown in Table 7:

TABLE 7

As can be seen from table 7:

when the combined use of the orlistat and the luteolin is carried out and the mass ratio of the orlistat to the luteolin is 0.1:10, the combined use coefficient CI is smaller than 1, the synergistic effect is shown, and the combined use index mean (CI _avg ) 0.82. When the mass ratio of the two is 0.1:8, the GI is that ₅₀ 、GI ₇₅ And GI ₉₀ Are smaller than 0.60, which shows stronger synergistic effect when the mass ratio of the two is 0.1:8, and the mean value of the combined drug index (CI) _avg ) 0.54. When the mass ratio of the two is 0.2:8, the combined drug coefficient CI is smaller than 1, the synergistic effect is shown, and the combined drug index mean (CI _avg ) 0.82.

When the mass ratio of orlistat to quercetin is 0.1:4, the combination drug coefficient CI is about 1, the superposition effect is shown, and the combination drug index mean (CI _avg ) 1.10. When the mass ratio of the two is 0.1:2, the GI is that ₅₀ 、GI ₇₅ And GI ₉₀ Are smaller than 0.70, which shows stronger synergistic effect when the mass ratio of the two is 0.1:2, and the mean value of the combined drug indexes (CI _avg ) 0.66. When the mass ratio of the two is 0.2:2, the combination drug coefficient CI is about 1, the superposition effect is shown, and the combination drug index mean value (CI _avg ) 0.99.

When the combined use of the orlistat and the dihydroquercetin is that the mass ratio of the orlistat to the dihydroquercetin is 0.1:70, the combined use coefficient CI is smaller than 1, which shows a synergistic effect,combination index mean (CI) _avg ) 0.57. When the mass ratio of the two is 0.1:60, the GI is that ₅₀ 、GI ₇₅ And GI ₉₀ Are smaller than 0.60, which shows stronger synergistic effect when the mass ratio of the two is 0.1:60, and the mean value of the combined drug index (CI) _avg ) 0.43. When the mass ratio of the two is 0.2:60, the combined drug coefficient CI is smaller than 1, the synergistic effect is shown, and the combined drug index mean (CI _avg ) 0.68.

When the combined use of orlistat and 3-O-methyl quercetin is carried out and the mass ratio of the orlistat to the 3-O-methyl quercetin is 0.1:60, the combined use coefficient CI is smaller than 1, the synergistic effect is shown, and the combined use index mean (CI _avg ) 0.60. When the mass ratio of the two is 0.1:50, the GI is that ₅₀ 、GI ₇₅ And GI ₉₀ Are smaller than 0.50, which shows stronger synergistic effect when the mass ratio of the two is 0.1:50, and the mean value of the combined drug indexes (CI) _avg ) 0.48. When the mass ratio of the two is 0.2:50, the combined drug coefficient CI is smaller than 1, the synergistic effect is shown, and the combined drug index mean (CI _avg ) 0.68.

When the combined use of orlistat and EGCG is carried out and the mass ratio of the orlistat to the EGCG is 0.1:30, the combined use coefficient CI is smaller than 1, the synergistic effect is shown, and the combined use index mean (CI _avg ) 0.60. When the mass ratio of the two is 0.1:20, the GI is that ₅₀ 、GI ₇₅ And GI ₉₀ Are smaller than 0.60, which shows stronger synergistic effect when the mass ratio of the two is 0.1:20, and the mean value of the combined drug indexes (CI _avg ) 0.51. When the mass ratio of the two is 0.2:20, the combined drug coefficient CI is smaller than 1, weak synergistic effect is shown, and the combined drug index mean (CI _avg ) 0.83.

Comparative example

In addition to the above examples, the present invention provides other cases where no synergistic effect with orlistat is formed during the screening of the combination of compositions, in which case compound a involves apigenin, morin, dihydromorin and fisetin. These case scenarios are only a part of the workload of the present invention at the time of creation. The following is shown:

first, IC of each monomer compound to pancreatic lipase was tested according to the method in the above example ₅₀ Values, as shown in table 8 below:

TABLE 8

Then, the monomer compound IC is obtained in proximity to each ₅₀ Values the pancreatic lipase inhibition was measured as shown in table 9:

TABLE 9

Compounds of formula (I)	Mass concentration (μg/mL)	Inhibition rate
			Apigenin
	20	45.1±1.5％
			Mulberry pigment	4	52.7±3.1％
Dihydromorin pigment	30	52.8±1.8％
			Fisetin	6	53.5±3.2％

The pancreatic lipase inhibition rates of the compositions were measured by combining the above-mentioned monomer compounds with orlistat, as shown in table 10 below.

Table 10

As can be seen from table 10, each of the above-mentioned monomer compounds, when combined with orlistat, exhibited a composition inhibition directly lower than that of the individual compounds, and the composition actually exhibited antagonism, without synergy.

In addition, the invention also tests the pancreatic lipase inhibition rates of luteolin and quercetin, dihydroquercetin, 3-O-methyl quercetin and EGCG in mass ratios of 8:2, 8:60, 8:50 and 8:20 respectively, and the results show that the inhibition rates of the composition are respectively 41.3+/-1.1%, 44.8+/-2.2%, 43.1+/-2.1% and 41.3+/-2.4%, which are lower than the inhibition rates of luteolin, quercetin, dihydroquercetin, 3-O-methyl quercetin and EGCG on pancreatic lipase alone (respectively 50.2+/-2.4%, 43.1+/-1.3%, 47.2+/-1.1%, 45.1+/-1.4% and 45.2+/-2.2%), which shows that the actual antagonism exists between the luteolin and the quercetin, the dihydroquercetin, the 3-O-methyl quercetin and the EGCG. The invention also tests the inhibition rate of the pancreatic lipase of quercetin and dihydroquercetin, 3-O-methyl quercetin and EGCG respectively at mass concentration ratios of 2:60, 2:50 and 2:20, and the results show that the inhibition rates of the composition are respectively 41.5+/-2.4%, 44.1+/-1.1% and 40.8+/-2.3% which are lower than the inhibition rates of the individual quercetin, dihydroquercetin, 3-O-methyl quercetin and EGCG on the pancreatic lipase (respectively 43.1+/-1.3%, 47.2+/-1.1%, 45.1+/-1.4% and 45.2+/-2.2%), thus indicating that the actual antagonism exists between the quercetin and the dihydroquercetin and the 3-O-methyl quercetin and between the EGCG. The invention also tests the pancreatic lipase inhibition rates of the dihydroquercetin, the 3-O-methyl quercetin and the EGCG respectively at mass concentration ratios of 60:50 and 60:20, and the results show that the inhibition rates of the composition are respectively 41.6+/-1.5% and 42.5+/-1.3%, which are lower than the inhibition rates of the dihydroquercetin, the 3-O-methyl quercetin and the EGCG on pancreatic lipase independently (respectively 47.2+/-1.1%, 45.1+/-1.4% and 45.2+/-2.2%), which indicates that antagonism exists between the dihydroquercetin, the 3-O-methyl quercetin and the EGCG. The invention also tests the pancreatic lipase inhibition rates of 3-O-methyl quercetin and EGCG respectively at the mass concentration ratio of 50:20, and the results show that the inhibition rates of the composition are respectively 39.8+/-1.3 percent, which are lower than the inhibition rates of 3-O-methyl quercetin and EGCG respectively on pancreatic lipase (45.1+/-1.4 percent and 45.2+/-2.2 percent respectively), thus indicating that antagonism exists between 3-O-methyl quercetin and EGCG.

The above description is only a preferred embodiment of the present invention, and is not intended to limit the invention in any way, and any person skilled in the art may make modifications or alterations to the disclosed technical content to the equivalent embodiments. However, any simple modification, equivalent variation and variation of the above embodiments according to the technical substance of the present invention still fall within the protection scope of the technical solution of the present invention.

Claims

1. An orlistat composition comprising orlistat and compound a; wherein, the compound A is selected from luteolin, quercetin, dihydroquercetin, 3-O-methyl quercetin or EGCG;

2. Orlistat composition according to claim 1, wherein the mass ratio of orlistat to luteolin is 0.1:8; the mass ratio of the orlistat to the quercetin is 0.1:2; the mass ratio of the orlistat to the dihydroquercetin is 0.1:60; the mass ratio of the orlistat to the 3-O-methyl quercetin is 0.1:50; the mass of the orlistat and the EGCG is 0.1:20.

3. Use of an orlistat composition according to claim 1 or 2 for the manufacture of a medicament with synergistic pancreatic lipase inhibition.

4. Use of an orlistat composition according to claim 1 or 2 for the manufacture of a medicament for the treatment of obesity.

5. A medicament having a synergistic pancreatic lipase inhibitory effect, which comprises the orlistat composition according to claim 1 or 2.

6. The medicament of claim 5, further comprising a pharmaceutically acceptable carrier, solvent, diluent, excipient, or other medium.

7. The medicament according to claim 5, wherein the dosage form of the medicament is selected from the group consisting of powders, granules, capsules, injections, oral liquids and tablets.