Synergistic antioxidant composition and its application

Abstract

The present invention discloses a synergistic antioxidant composition and its application, and belongs to the technical field of natural medicines. The composition of the present invention is composed of isorhamnetin and compound A, or kaempferol and EGCG; wherein compound A is selected from quercetin, luteolin or EGCG. The composition of the present invention has significant antioxidant activity, and thus has application value in the prevention or treatment of chronic diseases caused by oxidative stress. In addition, there is a synergistic effect between the components in the composition, and the combination thereof can greatly reduce the dosage of each component when used alone, and reduce the toxic and side effects of the active ingredients. Therefore, when achieving the corresponding antioxidant effect, the combination of active ingredients can effectively reduce the occurrence of drug resistance in the body, and has a good application prospect.

Description

Technical Field

[0001] The invention belongs to the technical field of natural medicines, and in particular relates to a synergistic antioxidant composition and application thereof. Background Art

[0002] Intracellular reactive oxygen species (ROS) are the main regulatory factors for maintaining intracellular homeostasis and mediating physiological and pathological signal transduction. Numerous studies have shown that oxidative stress can cause oxidative damage to biological macromolecules such as proteins, carbohydrates, lipids and DNA. This oxidative damage is often closely related to aging, cancer, cardiovascular disease and neurodegenerative diseases. Therefore, regulating intracellular ROS levels and maintaining intracellular homeostasis are crucial for maintaining the health of the body. The exogenous sources of antioxidants are mainly fruits, vegetables, herbs and spices.

[0003] Flavonoids are an important class of plant secondary metabolites, widely found in foods such as fruits, vegetables, tea, soy products and natural plants, and are the most abundant phenolic substances in the human diet. Due to the diversity of their molecular structures, flavonoids have a variety of physiological and pharmacological activities, such as antioxidant, anti-cancer, anti-inflammatory, hypoglycemic and antihypertensive, immunomodulatory, etc., and have the characteristics of low toxicity and small side effects. However, the long-term use of a single active substance is prone to drug resistance and has a high dependence on dosage, which will cause harm to the body over time.

[0004] The combination of active molecules can improve the effect, reduce the dosage, reduce toxic side effects, and avoid or delay the development of resistance to a certain extent. However, there are many types of flavonoids in nature, and the number of flavonoids is huge. At present, there are few examples of synergistic antioxidant combinations of flavonoid compounds. Therefore, it is of practical significance to develop a series of flavonoid compositions with synergistic antioxidant effects. Summary of the invention

[0005] The present invention provides a composition consisting of isorhamnetin and compound A, or consisting of kaempferol and EGCG; wherein compound A is selected from quercetin, luteolin or EGCG.

[0006] In the above composition, when compound A is quercetin, the mass ratio of isorhamnetin to quercetin is 4:4 to 5:3; when compound A is luteolin, the mass ratio of isorhamnetin to luteolin is 5:5 to 4:6; when compound A is EGCG, the mass ratio of isorhamnetin to EGCG is 4:7 to 5:6. In the above composition, the mass ratio of kaempferol to EGCG is 1.5:7 to 2:6.

[0007] The above composition has a significant antioxidant effect. Based on this, the present invention provides the use of the above composition in the preparation of medicines, health products and / or foods with antioxidant effects.

[0008] In the above composition, when the mass ratio of isorhamnetin to quercetin is 4:3-5:3, there is a synergistic antioxidant effect between the two; when the mass ratio of isorhamnetin to luteolin is 5:5-4:5, there is a synergistic antioxidant effect between the two; when the mass ratio of isorhamnetin to EGCG is 4:7-4:6, there is a synergistic antioxidant effect between the two; when the mass ratio of kaempferol to EGCG is 1.5:6-2:6, there is a synergistic antioxidant effect between the two. Based on this, in a specific embodiment, the composition provided by the present invention is composed of isorhamnetin and quercetin in a mass ratio of 4:3-.5:3; or, isorhamnetin and luteolin in a mass ratio of 5:5-4:5; or, isorhamnetin and EGCG in a mass ratio of 4:7-4:6; or, kaempferol and EGCG in a mass ratio of 1.5:6-2:6.

[0009] In a specific embodiment, the composition of the present invention is composed of isorhamnetin and quercetin in a mass ratio of 4:3; or, isorhamnetin and luteolin in a mass ratio of 4:5; or, isorhamnetin and EGCG in a mass ratio of 4:6; or, kaempferol and EGCG in a mass ratio of 1.5:6.

[0010] According to the above content, a medicine, health product and / or food with synergistic antioxidant effect can be provided accordingly, and the medicine, health product and / or food contains the above composition. In the present invention, the medicine 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.

[0011] The beneficial effects of the present invention are:

[0012] The composition of the present invention has significant antioxidant activity, and thus has application value in the prevention or treatment of chronic diseases caused by oxidative stress. In addition, there is a synergistic effect between the components in the composition, and the combined use thereof can greatly reduce the dosage of each component when used alone, and reduce the toxic and side effects of the active ingredients. Therefore, when achieving the corresponding antioxidant effect, the combined use of the active ingredients can effectively reduce the occurrence of drug resistance in the body, and has a good application prospect. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 The Fa-CI trend diagram of the antioxidant activity of the combination of isorhamnetin and quercetin (4:3);

[0014] Figure 2 The Fa-CI trend diagram of the antioxidant activity of the combination of isorhamnetin and luteolin (4:5);

[0015] Figure 3 This is the Fa-CI trend diagram of the antioxidant activity of the combination of isorhamnetin and EGCG (4:6);

[0016] Figure 4 This is the Fa-CI trend diagram of the antioxidant effect of the combination of kaempferol and EGCG (1.5:6). DETAILED DESCRIPTION

[0017] Isorhamnetin, molecular formula: C 16 H 12 O7; molecular weight: 316.26; CAS registration number: 480-19-3, structural formula:

[0018]

[0019] Quercetin, molecular formula is C 15 H 10 O7; molecular weight: 302.236; CAS registration number: 117-39-5, structural formula:

[0020]

[0021] Luteolin, molecular formula is C 15 H 10 O6; molecular weight: 286.24; CAS registration number: 491-70-3, structural formula:

[0022]

[0023] EGCG, molecular formula is C 22 H 18 O 11 ; Molecular weight: 458.38; CAS registration number: 989-51-5, structural formula:

[0024]

[0025] Kaempferol, molecular formula is C 15 H 10 O6; molecular weight: 286.24; CAS registration number: 520-18-3, structural formula:

[0026]

[0027] 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 intended to illustrate the present invention, and are not intended to limit the scope of the present invention in any form.

[0028] Example 1

[0029] The method for measuring antioxidant activity in cells, namely the CAA method, is as follows:

[0030] 1. Prepare drug solution

[0031] The test compound was prepared into a 10 mg / mL stock solution from dimethyl sulfoxide (DMSO); 9.47 mg DCFH-DA was dissolved in 1 mL methanol solution to prepare a 20 mM DCFH-DA stock solution; 542 mg ABAP was dissolved in 10 mL deionized water to prepare a 200 mM ABAP stock solution.

[0032] 2. Cell culture

[0033] HepG2 cells were cultured in MEM medium containing 10% fetal bovine serum and 1% double antibody (100×) in an incubator at 37° C., 5% CO 2 , and 95% humidity, and the medium was replaced every 1 to 2 days.

[0034] 3. Operation method

[0035] HepG2 cells were cultured at 5×10 4 The cells were inoculated in a 96-well cell culture plate at a density of 100 μM / well; the culture medium was removed after 24 hours of culture and washed once with PBS; the DCFH-DA probe culture medium containing the sample to be tested was added, wherein the final concentration of the DCFH-DA probe was 25 μM, and the incubation was continued for 1 hour; after 1 hour, the culture medium was removed, and 100 μL of PBS was added to each well for washing 3 times; then 100 μL of ABAP (diluted with Hanks solution) at a concentration of 600 μM was added, and the 96-well plate was placed in a multifunctional microplate reader for detection at a constant temperature of 37°C. The conditions for the microplate reader assay were: excitation wavelength 485 nm, emission wavelength 538 nm, oscillation 5 s, and then measured every 5 min for 60 min. In the experiment, each sample was repeated four times, and the experimental blank group was the fluorescence attenuation group with the addition of the probe DCFH-DA but without the addition of ABAP and antioxidants; the experimental positive control was the fluorescence attenuation group with the addition of the free radical exciter ABAP and the probe DCFH-DA, and the inhibition rate of the sample experimental dose on cell growth was below 10%.

[0036] 4. Calculation formula

[0037] CAA (unit) = 1-(∫SA / ∫CA)

[0038] In the above formula, ∫SA: the integrated area under the sample time-fluorescence value curve; ∫CA: the integrated area under the control time-fluorescence value curve. EC 50 : Calculated according to the median effect principle of log(fa / fu) / log(dose); fa: CAA unit; fu: 1-CAA unit.

[0039] The test subjects include isorhamnetin, quercetin, luteolin, EGCG, kaempferol, and combinations consisting of isorhamnetin and quercetin, isorhamnetin and luteolin, isorhamnetin and EGCG, and kaempferol and EGCG in different mass ratios.

[0040] The experimental instruments and reagents included: ABAP [2, 2-Azobis (2-amidinopropane) dihydrochloride solution], DCFH-DA (2′, 7′-Dichlorfluorescin diacetate), and sodium fluorescein were purchased from Sigma; isorhamnetin, quercetin, luteolin, EGCG, and kaempferol were purchased from Beijing Solebao; HepG2 cells (Kunming Institute of Zoology, Chinese Academy of Sciences), MEM culture medium (Gibco), Hanks balanced salt solution (HBSS), fetal bovine serum (FBS, Gibco), double antibody (Gibco), and trypsin (Gibco); Millipore Simplicity water purification system (Millipore, France), CO2 constant temperature incubator (Forma 371, Thermo Fisher), and microplate reader TECAN infinite M200 PRO (Teacan Group Ltd., Switzerland).

[0041] Specifically, isorhamnetin, quercetin, luteolin, EGCG, and kaempferol were used as test objects, and the cell antioxidant activity of each test object was tested using the above method. The half inhibitory concentration EC 50 Value representation.

[0042] The concentration gradient settings of each test object were as follows: isorhamnetin: 5μg / mL, 2.5μg / mL, 1.25μg / mL, 0.625μg / mL; quercetin: 5μg / mL, 2.5μg / mL, 1.25μg / mL, 0.625μg / mL; luteolin: 5μg / mL, 2.5μg / mL, 1.25μg / mL, 0.625μg / mL; EGCG: 8μg / mL, 4μg / mL, 2μg / mL, 1μg / mL; kaempferol: 2μg / mL, 1μg / mL, 0.5μg / mL, 0.25μg / mL.

[0043] The test results are shown in Table 1 below.

[0044] Table 1

[0045]

[0046] The data are derived from the results of three independent experiments and are expressed as "mean ± standard deviation".

[0047] Example 2

[0048] The combination of isorhamnetin and quercetin was used as the test object, and the cell antioxidant activity of each test object was tested using the method in Example 1. The half inhibitory concentration EC 50 Value representation.

[0049] The specific test objects are: isorhamnetin and quercetin (4:3), isorhamnetin and quercetin (5:3), isorhamnetin and quercetin (4:4). The above ratios are all mass ratios.

[0050] The concentration gradient settings of each test object are as follows: isorhamnetin and quercetin (4:3): (4+3) μg / mL, (2+1.5) μg / mL, (1+0.75) μg / mL, (0.5+0.375) μg / mL; isorhamnetin and quercetin (5:3): (5+3) μg / mL, (2.5+1.5) μg / mL, (1.25+0.75) μg / mL, (0.625+0.375) μg / mL; isorhamnetin and quercetin (4:4): (4+4) μg / mL, (2+2) μg / mL, (1+1) μg / mL, (0.5+0.5) μg / mL.

[0051] Principles for setting concentration gradient: Taking isorhamnetin and quercetin (4:3) as an example, the initial concentration of the composition is set to 7μg / mL, that is, the initial concentration of isorhamnetin in the composition sample solution is 4μg / mL, and the initial concentration of quercetin is 3μg / mL. On the basis of the initial concentration of 7μg / mL, the composition sample solution is diluted 2 times in sequence, namely 7μg / mL, 3.5μg / mL, 1.75μg / mL, and 0.875μg / mL. Similarly, in the composition of isorhamnetin and quercetin (5:3), the initial concentration of the composition is set to 8μg / mL, and it is diluted 2 times in sequence to form a concentration gradient. Other compositions also follow this principle.

[0052] The test results are shown in Table 2.

[0053] Table 2

[0054]

[0055] The data are derived from the results of three independent experiments and are expressed as "mean ± standard deviation".

[0056] It can be seen from Tables 1 and 2 that the combined use of isorhamnetin and quercetin can significantly reduce the dosage of each component when used alone and improve the antioxidant activity when the mass ratio of the two is 4:3, and the effect is more obvious.

[0057] Example 3

[0058] The combination of isorhamnetin and luteolin was used as the test object, and the cell antioxidant activity of each test object was tested using the method in Example 1. The half inhibitory concentration EC 50 Value representation.

[0059] The specific test objects are: isorhamnetin and luteolin (4:5), isorhamnetin and luteolin (5:5), isorhamnetin and luteolin (4:6). The above ratios are all mass ratios.

[0060] The concentration gradient settings of each test object were as follows: isorhamnetin and luteolin (4∶5): (4+5) μg / mL, (2+2.5) μg / mL, (1+1.25) μg / mL, (0.5+0.625) μg / mL; isorhamnetin and luteolin (5∶5): (5+5) μg / mL, (2.5+2.5) μg / mL, (1.25+1.25) μg / mL, (0.625+0.625) μg / mL; isorhamnetin and luteolin (4∶6): (4+6) μg / mL, (2+3) μg / mL, (1+1.5) μg / mL, (0.5+0.75) μg / mL.

[0061] Principles for setting concentration gradient: Taking isorhamnetin and luteolin (4:5) as an example, the initial concentration of the composition is set to 9 μg / mL, that is, the initial concentration of isorhamnetin in the composition sample solution is 4 μg / mL, and the initial concentration of luteolin is 5 μg / mL. On the basis of the initial concentration of 9 μg / mL, the composition sample solution is diluted 2 times in sequence, namely 9 μg / mL, 4.5 μg / mL, 2.25 μg / mL, and 1.125 μg / mL. Similarly, in the composition of isorhamnetin and luteolin (5:5), the initial concentration of the composition is set to 10 μg / mL, and it is diluted 2 times in sequence to form a concentration gradient. Other compositions also follow this principle.

[0062] The test results are shown in Table 3.

[0063] Table 3

[0064]

[0065] The data are derived from the results of three independent experiments and are expressed as "mean ± standard deviation".

[0066] It can be seen from Table 1 and Table 3 that the combined use of isorhamnetin and luteolin can significantly reduce the dosage of each component when used alone and improve the antioxidant activity when the mass ratio of the two is 4:5, especially when the mass ratio of the two is 4:5.

[0067] Example 4

[0068] The combination of isorhamnetin and EGCG was used as the test object, and the cell antioxidant activity of each test object was tested using the method in Example 1. The half inhibitory concentration EC 50 Value representation.

[0069] The specific test objects are: isorhamnetin and EGCG (4:6), isorhamnetin and EGCG (5:6), isorhamnetin and EGCG (4:7). The above ratios are all mass ratios.

[0070] The concentration gradient settings of each test object were as follows: isorhamnetin and EGCG (4∶6): (4+6) μg / mL, (2+3) μg / mL, (1+1.5) μg / mL, (0.5+0.75) μg / mL; isorhamnetin and EGCG (5∶6): (5+6) μg / mL, (2.5+3) μg / mL, (1.25+1.5) μg / mL, (0.625+0.75) μg / mL; isorhamnetin and EGCG (4∶7): (4+7) μg / mL, (2+3.5) μg / mL, (1+1.75) μg / mL, (0.5+0.875) μg / mL.

[0071] Principles for setting concentration gradient: Taking isorhamnetin and EGCG (4:6) as an example, the initial concentration of the composition is set to 10 μg / mL, that is, the initial concentration of isorhamnetin in the composition sample solution is 4 μg / mL, and the initial concentration of EGCG is 6 μg / mL. On the basis of the initial concentration of 10 μg / mL, the composition sample solution is diluted 2 times in sequence, namely 10 μg / mL, 5 μg / mL, 2.5 μg / mL, and 1.25 μg / mL. Similarly, in the composition of isorhamnetin and EGCG (5:6), the initial concentration of the composition is set to 11 μg / mL, and it is diluted 2 times in sequence to form a concentration gradient. Other compositions also follow this principle.

[0072] The test results are shown in Table 4.

[0073] Table 4

[0074]

[0075] The data are derived from the results of three independent experiments and are expressed as “mean ± SD”.

[0076] It can be seen from Table 1 and Table 4 that the combined use of isorhamnetin and EGCG can significantly reduce the dosage of each component when used alone and improve the antioxidant activity when the mass ratio of the two is 4:6, especially when the mass ratio of the two is 4:6.

[0077] Example 5

[0078] The combination of kaempferol and EGCG was used as the test object, and the cell antioxidant activity of each test object was tested using the method in Example 1. The half inhibitory concentration EC 50 Value representation.

[0079] The specific test objects are: kaempferol and EGCG (1.5:6), kaempferol and EGCG (2:6), kaempferol and EGCG (1.5:7). The above ratios are all mass ratios.

[0080] The concentration gradient settings for each test object were as follows: kaempferol and EGCG (1.5∶6): (1.5+6) μg / mL, (0.75+3) μg / mL, (0.375+1.5) μg / mL, (0.1875+0.75) μg / mL; kaempferol and EGCG (2∶6): (2+6) μg / mL, (1+3) μg / mL, (0.5+1.5) μg / mL, (0.25+0.75) μg / mL; kaempferol and EGCG (1.5∶7): (1.5+7) μg / mL, (0.75+3.5) μg / mL, (0.375+1.75) μg / mL, (0.1875+0.875) μg / mL.

[0081] Principles for setting concentration gradient: Taking kaempferol and EGCG (1.5:6) as an example, the initial concentration of the composition is set to 7.5 μg / mL, that is, the initial concentration of kaempferol in the composition sample solution is 1.5 μg / mL, and the initial concentration of EGCG is 6 μg / mL. On the basis of the initial concentration of 7.5 μg / mL, the composition sample solution is diluted 2 times in sequence, namely 7.5 μg / mL, 3.75 μg / mL, 1.875 μg / mL, and 0.9375 μg / mL. Similarly, in the composition of kaempferol and EGCG (2:6), the initial concentration of the composition is set to 8 μg / mL, and it is diluted 2 times in sequence to form a concentration gradient. Other compositions also follow this principle.

[0082] The test results are shown in Table 5.

[0083] Table 5

[0084]

[0085] The data are derived from the results of three independent experiments and are expressed as "mean ± standard deviation".

[0086] It can be seen from Table 1 and Table 5 that the combined use of kaempferol and EGCG can significantly reduce the dosage of each component when used alone and improve the antioxidant activity when the mass ratio of the two is 1.5:6, especially when the mass ratio of the two is 1.5:6.

[0087] Determination of drug combination coefficient

[0088] The combination coefficient (CI) of the compositions in Example 2, Example 3, Example 4 and Example 5 was measured, the CI value was calculated according to the software CompuSyn, and the synergistic effect between the drugs was evaluated.

[0089] 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.

[0090] Taking isorhamnetin and quercetin (4:3) in Example 2 as an example, the initial concentration of the composition is set to 7 μg / mL, the initial concentration of isorhamnetin is 4 μg / mL, and the initial concentration of quercetin is 3 μg / mL; therefore, the gradient concentration setting of isorhamnetin is to dilute it 2-fold on the basis of the initial concentration of 4 μg / mL, and to be 4 μg / mL, 2 μg / mL, 1 μg / mL, and 0.5 μg / mL in sequence; the gradient concentration setting of quercetin is to dilute it 2-fold on the basis of the initial concentration of 3 μg / mL, and to be 3 μg / mL, 1.5 μg / mL, 0.75 μg / mL, and 0.375 μg / mL in sequence.

[0091] The inhibition rates of isorhamnetin, quercetin, isorhamnetin and quercetin (4:3) at different concentration gradients were statistically analyzed, and the results are as follows:

[0092] The inhibition rates of isorhamnetin under the concentration gradient were 13.3±0.1%, 21.4±1.1%, 28.4±1.5%, and 44.6±2.2% (from low concentration to high concentration), respectively; the inhibition rates of quercetin under the concentration gradient were 14.9±1.2%, 20.5±1.2%, 29.5±1.1%, and 49.2±2.2% (from low concentration to high concentration), respectively; the inhibition rates of isorhamnetin and quercetin (4:3) under the concentration gradient were 28.4±1.2%, 48.3±1.4%, 61.4±1.3%, and 75.5±2.2% (from low concentration to high concentration), respectively.

[0093] The above data were processed using CompuSyn software to obtain the CI values ​​of isorhamnetin and quercetin in the isorhamnetin and quercetin (4:3) composition, as shown in Table 6. Thus, the Fa-CI trend of the antioxidant activity of the isorhamnetin and quercetin (4:3) composition can be obtained, as shown in Table 6. Figure 1 shown.

[0094] Taking isorhamnetin and luteolin (4:5) in Example 3 as an example, the initial concentration of the composition is set to 9 μg / mL, the initial concentration of isorhamnetin is 4 μg / mL, and the initial concentration of luteolin is 5 μg / mL; therefore, the gradient concentration setting of isorhamnetin is to dilute it 2-fold on the basis of the initial concentration of 4 μg / mL, and to be 4 μg / mL, 2 μg / mL, 1 μg / mL, and 0.5 μg / mL in sequence; the gradient concentration setting of luteolin is to dilute it 2-fold on the basis of the initial concentration of 5 μg / mL, and to be 5 μg / mL, 2.5 μg / mL, 1.25 μg / mL, and 0.625 μg / mL in sequence.

[0095] The inhibition rates of isorhamnetin, luteolin, isorhamnetin and luteolin (4:5) at different concentration gradients were statistically analyzed, and the results are as follows:

[0096] The inhibition rates of isorhamnetin under the concentration gradient were 13.3±0.1%, 21.4±1.1%, 28.4±1.5%, and 44.6±2.2% (from low concentration to high concentration), respectively; the inhibition rates of luteolin under the concentration gradient were 10.5±0.5%, 22.2±1.1%, 39.1±1.2%, and 58.2±1.3% (from low concentration to high concentration), respectively; the inhibition rates of isorhamnetin and luteolin (4:5) under the concentration gradient were 30.4±1.1%, 41.3±1.2%, 72.4±1.3%, and 85.5±2.2% (from low concentration to high concentration), respectively.

[0097] The above data were processed using CompuSyn software to obtain the CI values ​​of isorhamnetin and luteolin in the isorhamnetin and luteolin (4:5) composition, as shown in Table 6. Thus, the Fa-CI trend of the antioxidant activity of the isorhamnetin and luteolin (4:5) composition can be obtained, as shown in Table 6. Figure 2 shown.

[0098] Taking isorhamnetin and EGCG (4:6) in Example 4 as an example, the initial concentration of the composition is set to 10 μg / mL, the initial concentration of isorhamnetin is 4 μg / mL, and the initial concentration of EGCG is 6 μg / mL; therefore, the gradient concentration setting of isorhamnetin is to dilute it 2-fold on the basis of the initial concentration of 4 μg / mL, and to be 4 μg / mL, 2 μg / mL, 1 μg / mL, and 0.5 μg / mL in sequence; the gradient concentration setting of EGCG is to dilute it 2-fold on the basis of the initial concentration of 6 μg / mL, and to be 6 μg / mL, 3 μg / mL, 1.5 μg / mL, and 0.75 μg / mL in sequence.

[0099] The inhibition rates of isorhamnetin, EGCG, isorhamnetin and EGCG (4:6) at different concentration gradients were statistically analyzed, and the results are as follows:

[0100] The inhibition rates of isorhamnetin under the concentration gradient were 13.3±0.1%, 21.4±1.1%, 28.4±1.5%, and 44.6±2.2% (from low concentration to high concentration), respectively; the inhibition rates of EGCG under the concentration gradient were 7.5±0.2%, 13.5±1.3%, 25.2±1.2%, and 44.2±1.1% (from low concentration to high concentration), respectively; the inhibition rates of isorhamnetin and EGCG (4:6) under the concentration gradient were 22.4±1.3%, 34.3±1.1%, 48.4±1.4%, and 65.5±2.1% (from low concentration to high concentration).

[0101] The above data were processed using CompuSyn software to obtain the CI values ​​of isorhamnetin and EGCG in the isorhamnetin and EGCG (4:6) composition, as shown in Table 6. Thus, the Fa-CI trend of the antioxidant activity of the isorhamnetin and EGCG (4:6) composition can be obtained, as shown in Table 6. Figure 3 shown.

[0102] Taking kaempferol and EGCG (1.5:6) in Example 5 as an example, the initial concentration of the composition is set to 7.5 μg / mL, the initial concentration of kaempferol is 1.5 μg / mL, and the initial concentration of EGCG is 6 μg / mL; therefore, the gradient concentration setting of kaempferol is based on the initial concentration of 1.5 μg / mL, and is successively diluted by 2 times, namely 1.5 μg / mL, 0.75 μg / mL, 0.375 μg / mL, and 0.1875 μg / mL; the gradient concentration setting of EGCG is based on the initial concentration of 6 μg / mL, and is successively diluted by 2 times, namely 6 μg / mL, 3 μg / mL, 1.5 μg / mL, and 0.75 μg / mL.

[0103] The inhibition rates of kaempferol, EGCG, kaempferol and EGCG (1.5:6) at different concentration gradients were statistically analyzed, and the results were as follows:

[0104] The inhibition rates of kaempferol under the concentration gradient were 6.3±0.2%, 15.4±1.4%, 28.4±1.3%, and 40.6±2.1% (from low concentration to high concentration), respectively; the inhibition rates of EGCG under the concentration gradient were 7.5±0.2%, 13.5±1.3%, 25.2±1.2%, and 44.2±1.1% (from low concentration to high concentration), respectively; the inhibition rates of kaempferol and EGCG (1.5:6) under the concentration gradient were 20.4±1.4%, 35.3±1.6%, 55.4±1.7%, and 68.5±2.6% (from low concentration to high concentration), respectively.

[0105] CompuSyn software was used to process the above data to obtain the CI values ​​of kaempferol and EGCG in the composition of kaempferol and EGCG (1.5:6), as shown in Table 6. Thus, the Fa-CI trend of the antioxidant activity of the composition of kaempferol and EGCG (1.5:6) can be obtained, as shown in Table 6. Figure 4 shown.

[0106] Similarly, for other compositions, including isorhamnetin and quercetin (4:4), isorhamnetin and quercetin (5:3), isorhamnetin and luteolin (4:6), isorhamnetin and luteolin (5:5), isorhamnetin and EGCG (4:7), isorhamnetin and EGCG (5:6), kaempferol and EGCG (1.5:7), and kaempferol and EGCG (2:6), the concentration gradient setting of each monomer compound and the calculation of CI values ​​are all based on the above principles.

[0107] The measurement results are shown in Table 6:

[0108] Table 6

[0109]

[0110]

[0111] The data are derived from the results of three independent experiments and are expressed as "mean ± standard deviation".

[0112] From Table 6, we can see that:

[0113] When the mass ratio of isorhamnetin and quercetin was 4:4, the combined drug index CI was about 1, showing an additive effect. avg ) is 0.92. When the mass ratio of the two is 4:3, GI 50 GI 75 and GI 90 were all less than 0.50, indicating that the two showed a strong synergistic effect when the mass ratio was 4:3. The mean value of the combined drug index (CI avg ) was 0.37. When the mass ratio of the two was 5∶3, the combined drug index CI was less than 0.8, showing a synergistic effect. The combined drug index mean (CI avg ) is 0.69.

[0114] When the mass ratio of isorhamnetin and luteolin was 4:6, the combined drug index CI was about 1, showing an additive effect. avg ) is 0.94. When the mass ratio of the two is 4:5, GI 50 GI 75 and GI 90 were all less than 0.60, indicating that the two showed a strong synergistic effect when the mass ratio was 4:5. The mean value of the combined drug index (CI avg ) was 0.54. When the mass ratio of the two was 5:5, a weak synergistic effect was shown, and the mean value of the combined drug index (CI avg ) is 0.86.

[0115] The combined use of isorhamnetin and EGCG, when the mass ratio of the two was 4:7, had a combined drug coefficient CI less than 1, showing a weak synergistic effect. avg ) is 0.86. When the mass ratio of the two is 4:6, GI 50 GI 75 and GI 90 were all less than 0.80, indicating that the two showed synergistic effects when the mass ratio was 4∶6. avg ) was 0.74. When the mass ratio of the two was 5:6, the combined drug coefficient CI was greater than 1, showing an antagonistic effect.

[0116] When the mass ratio of kaempferol and EGCG was 1.5:7, the combined medication coefficient CI was greater than 1, showing an antagonistic effect. 50 GI 75 and GI 90 were all less than 0.60, indicating that the two showed a strong synergistic effect when the mass ratio was 1.5∶6. The mean value of the combined drug index (CI avg ) was 0.54. When the mass ratio of the two was 2:6, a weak synergistic effect was shown, and the mean value of the combined drug index (CI avg ) is 0.85.

[0117] Comparative Example

[0118] In addition to the above-mentioned implementation cases, the present invention also provides other cases in which synergy cannot be formed with isorhamnetin during the screening process of the combination of the composition. In these cases, compound A involves rhamnetin, morin, 3-O methylquercetin, isorhamnetin and myricetin. There are also other cases in which synergy cannot be formed with kaempferol. In these cases, the compounds that cannot form synergy with kaempferol involve baicalein, tannin, isorhamnetin and herbicide. These cases are only part of the workload of the present invention at the time of creation. As shown below:

[0119] First, the EC of each monomer compound for cell antioxidant activity was tested according to the method in the above example. 50 Values ​​are shown in Table 7 below:

[0120] Table 7

[0121]

[0122]

[0123] Then, take the value close to the EC of each monomer compound. 50The antioxidant inhibition rate was determined based on the values ​​of the values, as shown in Table 8:

[0124] Table 8

[0125] Compound Mass concentration (μg / mL) Inhibition rate Rhamnetin 2.5 52.2±1.4％ Morin 2.5 51.7±2.1％ 3-O-Methylquercetin 6 48.8±1.2％ Isorientin 80 50.5±3.2％ Myricetin 15 50.2±2.2％ Baicalin 5 46.2±1.7％ Xanthophyllotoxin 6 48.2±1.4％ Isorhamnetin 4 44.6±2.2％ Herbaceous 15 51.2±1.5％ Kaempferol 1.5 40.6±2.1％

[0126] The above monomer compounds were combined with isorhamnetin and kaempferol to measure the antioxidant activity of the composition, as shown in the following Table 9:

[0127] Table 9

[0128]

[0129]

[0130] It can be seen from Table 10 that after the above monomer compounds are combined with isorhamnetin and kaempferol, the inhibition rate of the combination is directly lower than the inhibition rate of the single compound, and there is no synergistic effect.

[0131] 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 having an antioxidant effect, characterized in that: The invention is composed of kaempferol and EGCG; the mass ratio of kaempferol to EGCG is 1.5:

6.

2. Use of the composition according to claim 1 in the preparation of medicines, health products and / or foods with antioxidant effects.

3. A medicine, health product and / or food having an antioxidant effect, characterized in that: The active ingredient of the medicine, health product and / or food is the composition according to claim 1.

4. The drug according to claim 3, characterized in that The medicament further comprises a pharmaceutically acceptable carrier.

5. The drug according to claim 4, characterized in that The dosage form of the drug is selected from powder, granules, capsules, injections, oral liquids or tablets.

Images