Use of demethoxycurcumin and phloretin in the preparation of a drug for inhibiting beta-lactoglobulin glycosylation

Abstract

This invention belongs to the field of pharmacology and provides the application of demethoxycurcumin and phlorizin in the preparation of drugs that inhibit β-lactoglobulin glycosylation. The synergistic inhibitory effects and mechanisms of demethoxycurcumin and phlorizin on AGEs were systematically investigated. Fluorescence spectroscopy and carbonyl / thiol / amino group assays confirmed that demethoxycurcumin and phlorizin can synergistically inhibit β-lactoglobulin glycosylation at appropriate concentrations. Circular dichroism spectroscopy and other experiments showed that the combined use of the two can better alleviate protein secondary structure changes and aggregation. They competitively bind to the same glycosylation site, synergistically resist oxidation, thereby inhibiting AGEs formation, and synergistically reducing oxidative stress, cell damage, and apoptosis at the cellular level. Therefore, the combined application of these two drugs has potential therapeutic value in intervening in diabetic complications.

Description

Technical Field

[0001] This invention relates to the field of pharmacology, and more particularly to the application of demethoxycurcumin and phloretin in the preparation of drugs that inhibit β-lactoglobulin glycosylation. Background Technology

[0002] Advanced glycation end products (AGEs) are the final products of the Maillard reaction. Their accumulation in the body can lead to various chronic diseases, especially diabetes. Cardiovascular complications (CDV) are a leading cause of death in diabetic patients. The interaction between AGEs and AGEs receptors induces oxidative stress and apoptosis in endothelial cells, leading to endothelial cell dysfunction and accelerating the progression of diabetic vascular complications. Therefore, inhibiting the production of AGEs is particularly important for the prevention of CDV.

[0003] Current research has demonstrated that synthetic drugs, polyphenols, and polysaccharides can inhibit the formation of AGEs, but single inhibitors have limitations such as high toxicity, dose dependence, and drug resistance. The intake of exogenous AGEs directly increases the total AGEs load in the body's circulation and tissues. Dairy products are an important source of dietary protein and nutrition, and AGEs are easily formed during heat treatment processes such as pasteurization and spray drying. Lactoglobulin (BLG) is the main protein in bovine whey. Because it contains 15 lysine residues and one N-terminal α-amino group of its peptide chain, it is more susceptible to the Maillard reaction. Similarly, lactose (Lac), a naturally occurring reducing disaccharide in milk, is a major participant in the Maillard reaction in dairy systems. Glycosylation in dairy products leads to nutrient loss and increases the total AGEs load in the human body; therefore, inhibiting the formation of AGEs during dairy processing is particularly important. Summary of the Invention

[0004] The purpose of this invention is to provide the application of demethoxycurcumin and phlorizin in the preparation of drugs that inhibit β-lactoglobulin glycosylation. Demethoxycurcumin (DMC), extracted from the rhizome of the traditional herb turmeric, has been shown to effectively inhibit BLG glycosylation by occupying the glycosylation sites of BLG. Phlorizin (PHL), a dihydrochalcone flavonoid compound, is abundant in apples and strawberries and can effectively improve diabetes-related complications such as cardiomyopathy, hypertension, depression, memory impairment, delayed wound healing, and peripheral neuropathy. This indicates that both DMC and PHL can effectively inhibit AGEs formation and prevent diabetic complications; however, whether they have a synergistic inhibitory effect on AGEs and the mechanism thereof have not been studied, and whether they have a synergistic preventive effect on CDV remains a blank. Therefore, the synergistic inhibitory effect of DMC and PHL on AGEs and CDV warrants further investigation. Furthermore, the interaction between polyphenols and proteins is one of the important methods for studying their inhibitory mechanisms. Studying the interaction between DMC and PHL and BLG can provide a theoretical basis for understanding their inhibitory mechanism.

[0005] This study investigated the synergistic inhibitory effects of DMC and PHL on AGEs using fluorescence spectroscopy and carbonyl thiol content determination. The mechanism of their synergistic inhibition of AGEs was analyzed through multispectral analysis, molecular docking, and antioxidant assays. Furthermore, intracellular ROS detection, MTT assay, and apoptosis studies were used to investigate their synergistic improvement of AGEs-induced cell damage. This research lays a solid scientific foundation for the development of safe, efficient, and multi-component synergistic anti-glycation agents.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution: The present invention provides a functional composition comprising the following components: demethoxycurcumin and phloretin.

[0007] The present invention also provides the use of the described composition in the preparation of anti-glycation drugs.

[0008] Preferably, the concentration of demethoxycurcumin in the anti-glycation drug is 30-50 μM, the concentration of phloretin in the anti-glycation drug is 140-160 μM, and the anti-glycation drug is a drug that inhibits the formation of advanced glycation end products.

[0009] The present invention also provides the use of the described composition in the preparation of antioxidant drugs.

[0010] Preferably, the concentration of demethoxycurcumin in the antioxidant drug is 5-9 μM, and the concentration of phloretin in the antioxidant drug is 4-8 μM.

[0011] The beneficial effects of this invention are as follows: This invention systematically investigated the synergistic inhibitory effects and mechanisms of demethoxycurcumin (DMC) and phloretin (PHL) on advanced aging reactions (AGEs). Fluorescence spectroscopy and determination of carbonyl, thiol, and amino groups showed that DMC and PHL, both polyphenols, synergistically inhibited BLG glycosylation within a certain concentration range. Circular dichroism spectroscopy, scanning electron microscopy, and thiosulfate T experiments indicated that DMC and PHL effectively alleviated glycosylation-induced secondary structure changes and aggregation of BLG, with the combined polyphenols showing a more significant effect. Fluorescence quenching and molecular docking experiments showed that DMC and PHL competitively bind to the same glycosylation site in BLG; the simultaneous binding of the two polyphenols may lead to greater steric hindrance, preventing the reaction of lysine and lac to produce AGEs, thus achieving a synergistic inhibitory effect on AGEs. Free radical scavenging experiments showed that DMC and PHL synergistically inhibited AGE formation through antioxidant effects. Cellular experiments showed that AGEs can induce oxidative stress, reduce cell viability, and lead to apoptosis in human umbilical vein cell fusion cells (Ea.hy926). DMC and PHL not only reduce cell damage at its source by inhibiting AGEs production, but also directly inhibit AGEs-induced ROS generation and apoptosis, and exhibit synergistic effects when used in combination. Their combined application has potential therapeutic value in intervening in diabetic complications. Attached Figure Description

[0012] Figure 1 (A, B) Inhibition rate of AGEs by different concentrations of DMC or PHL, (C, D) Changes in SE value of AGEs inhibited by different concentrations of DMC and PHL; Figure 2 The relative amino content of BLG, AGEs, BLG+Lac+DMC, BLG+Lac+PHL, and BLG+Lac+PHL+DMC; Figure 3 CD spectra (A), secondary structure content (B), and particle size distribution (C) of BLG, AGEs, BLG+Lac+DMC, BLG+Lac+PHL, and BLG+Lac+PHL+DMC. Figure 4 SEM images of BLG(A), AGEs(B), BLG+Lac+DMC(C), BLG+Lac+PHL(D) and BLG+Lac+PHL+DMC(E); Figure 5 SDS-PAGE (A), ThT fluorescence spectra of BLG, AGEs, BLG+Lac+DMC, BLG+Lac+PHL and BLG+Lac+PHL+DMC (B); Figure 6The fluorescence spectra of the interaction between DMC(A,B) and PHL(C,D) and BLG(A,C) or AGEs(B,D) at 25℃ are shown. Figure 7 The fluorescence spectrum of the ternary system at 25℃ is shown below. Figure 8 The optimal docking results for DMC(A) and PHL(B) with BLG molecules were obtained. Figure 9 The optimal docking result for PHL and (DMC+BLG) molecules is shown in (A), and the optimal docking result for DMC and (PHL+BLG) molecules is shown in (B). Figure 10 Three-dimensional response surface plots of (A) the free radical scavenging capacity of DMC and PHL at different concentrations, and (B) the effect of DMC and PHL at different concentrations on SE. Figure 11 ROS fluorescence micrographs: BLG(A), AGEs(B), BLG+Lac+DMC(C), BLG+Lac+PHL(D), BLG+Lac+PHL+DMC(E), AGEs+DMC(F), AGEs+PHL(G), and AGEs+DMC+PHL(H). Figure 12 (A) The relative fluorescence intensity of ROS generated in Ea.hy926; (B) The in vitro cytotoxicity of Ea.hy926; Figure 13 Figure 1 shows apoptosis induced by BLG(A), AGEs(B), BLG+Lac+DMC(C), BLG+Lac+PHL(D), BLG+Lac+PHL+DMC(E), AGEs+DMC(F), AGEs+PHL(G) and AGEs+DMC+PHL(H). Detailed Implementation

[0013] The present invention provides a functional composition comprising the following components: demethoxycurcumin and phloretin.

[0014] The present invention also provides the use of the described composition in the preparation of anti-glycation drugs.

[0015] In this invention, the concentration of demethoxycurcumin in the anti-glycation drug is preferably 30-50 μM, more preferably 35-45 μM, and even more preferably 40 μM; the concentration of phloretin in the anti-glycation drug is preferably 140-160 μM, more preferably 145-155 μM, and even more preferably 150 μM; and the anti-glycation drug is preferably a drug that inhibits the formation of advanced glycation end products.

[0016] The present invention also provides the use of the described composition in the preparation of antioxidant drugs.

[0017] In this invention, the concentration of demethoxycurcumin in the antioxidant drug is preferably 5-9 μM, more preferably 7 μM, and the concentration of phloretin in the antioxidant drug is preferably 4-8 μM, more preferably 6 μM.

[0018] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0019] Example

[0020] 1. Experimental reagents

[0021] BLG, Lac, DMC, PHL, trichloroacetic acid (TCA, 20%, [w / v]), o-phthalaldehyde (OPA), 2,2'-hydrazine-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), and 2,2'-azobisisobutylamidine dihydrochloride (AAPH) were purchased from Yuanye Biotechnology Co., Ltd. (Shanghai, China). Thiazol blue (MTT) was purchased from Sigma Aldrich (Louis, USA). The Annexin V-FITC / PI apoptosis detection kit was purchased from Beyotime Biotechnology Co., Ltd. (Shanghai, China). Except for BLG and TCA, the purity of all samples was ≥98%. Human umbilical vein cell fusion cells (Ea.hy926) were provided by the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). DMC and PHL were dissolved in ethanol, and the final ethanol content in all samples did not exceed 2%.

[0022] 2. Preparation of AGEs and the synergistic inhibitory effect of DMC and PHL on AGEs

[0023] 2.1 Preparation of AGEs

[0024] Based on existing methods, a model for AGEs was constructed with slight modifications. BLG (0.1 mM) and Lac (150 mM) were mixed in 0.1 M phosphate-buffered saline (PBS, pH 7.4) and incubated at 70°C to obtain Lac-induced BLG glycosylation products. The concentration of BLG was then diluted to 20... After M, the sample was monitored at 360~600 nm using an F-7000 fluorescence spectrophotometer (Hitachi, Japan). ex The fluorescence intensity at 340 nm was used as a blank correction by subtracting the natural BLG fluorescence. The reaction was stopped when the fluorescence intensity reached saturation.

[0025] 2.2 Synergistic inhibitory effect of DMC and PHL on AGEs

[0026] BLG+Lac+DMC, BLG+Lac+PHL, and BLG+Lac+DMC+PHL were designated as positive groups, and the corresponding negative groups were BLG+DMC, BLG+PHL, and BLG+DMC+PHL. BLG+Lac served as the control group, and BLG was the blank control group. The concentrations of DMC were 30, 35, 40, 45, 50, and 55%. M, PHL concentrations were 100, 125, 150, 175, 200, and 225 μM, with a fixed DMC concentration of 40 μM. M, PHL are 100, 125, 150, 175, 200, 225 and 250 respectively. M. Fixed PHL concentration at 150 M, DMC are 25, 30, 35, 40, 45, 50 and 60 respectively. M. Experimental conditions were the same as in 2.1. The inhibition rate was calculated using Equation 1, and the synergistic coefficient (SE) between DMC and PHL was calculated using Equations 2 and 3.

[0027] Formula 1; F c , F b , F p and F n The fluorescence intensities of the control group, blank group, positive group, and negative group samples are represented respectively. The half-inhibitory concentrations (IC50) of DMC and PHL were calculated using CompuSyn software. 50 ).

[0028] Formula 2; in, IR 1 and IR 2 represents the inhibition rate of DMC and PHL, respectively.

[0029] Formula 3; SE<1, =1, and >1 indicate that the two polyphenols have antagonistic, additive, and synergistic effects, respectively.

[0030] 2.3 Study on carbonyl and thiol group content

[0031] Considering the single-component inhibition rates of DMC and PHL and the SE value, 40 was selected. M's DMC with 150 M's PHL, alone or in combination, inhibited AGEs to prepare reaction samples. The reaction samples were dialyzed against PBS (pH 7.4, 10 mM) at 4 °C for 48 h to remove free Lac. The obtained samples were lyophilized and stored at 20 °C for the determination of carbonyl content and subsequent experiments.

[0032] Carbonyl content was determined using the DNPH method. The sample solution (1.5 mg / mL, 1.2 mL) was mixed with DNPH (10 mM, 4.8 mL) dissolved in HCl (2.5 M) and incubated in the dark for 1 h. The mixture was placed in an ice bath, and TCA (6 mL) was added to terminate the reaction. The resulting solution was centrifuged (10,000 g, 4 °C) for 10 min to obtain a precipitate. The precipitate was then washed three times with 12 mL of ethanol / ethyl acetate (1:1, v:v). The precipitate was dissolved in guanidine hydrochloride (6 M, 3 mL), and a blank sample without DNPH was used. The absorbance at 370 nm was recorded using a UV-Vis spectrophotometer (U-3900H, Hitachi, Japan). Carbonyl content was determined based on a molar extinction coefficient of 22000 L·M⁻¹. -1 ·cm -1 calculate.

[0033] The free thiol content was determined using Ellman's method. The sample solution (1.5 mg / mL, 3 mL) was mixed with DTNB (4 mg / mL, 50 μL) and incubated at 25 °C for 30 min. The absorbance at 412 nm was recorded, and the result was determined using the molar extinction coefficient of DTNB, 13600 L·M⁻¹. - ¹·cm - ¹Calculate the thiol content.

[0034] 2.4 Determination of Amino Content

[0035] The free amino content was determined using the OPA method. OPA (40 mg) was dissolved in 1 mL of methanol, and then reacted with sodium dodecyl sulfate (2.5 mL, 20%, w / v) and β-mercaptoethanol (100 mg / mL). Mix 1 L of borax (0.1 M, 25 mL) and dilute with deionized water to 50 mL to prepare the OPA reagent. Add 3 mL of the OPA reagent to the sample solution (2 mg / mL, 150 mL) and dilute with deionized water to prepare the OPA reagent. The mixture (L) was incubated at 35°C for 2 min. The relative free amino content was calculated by comparing the absorbance of BLG, AGEs, and BLG+Lac+DMC / PHL / (DMC+PHL) at 340 nm.

[0036] 3. Conformational Change Analysis

[0037] 3.1 CD spectral studies

[0038] CD spectra of BLG, AGEs, and BLG+Lac+DMC / PHL / (DMC+PHL) were measured using a Jasco J-810 spectropolarimeter (Tokyo, Japan), scanning in the range of 190–240 nm. The molar concentration of all samples was kept constant at 15. M. Quantitative analysis of CD spectra was performed using DichroWeb-Online CD Analysis.

[0039] 3.2 DLS Research

[0040] The hydrodynamic diameters of BLG, AGEs, and BLG+Lac+DMC / PHL / (DMC+PHL) were determined using a Zetasizer Nano ZS (Malvern, UK) at 25°C. D h The laser was used to detect the zeta potential and the wavelength of the laser. A 4 mW He-Ne laser (wavelength 633 nm) was employed at a detection angle of 173°. All samples were at a concentration of 10⁻⁶. M, with a length of 0.22 Measurement after filtration through a filter membrane.

[0041] 4. Aggregation Research

[0042] 4.1 SEM Research

[0043] Microstructure images of BLG, AGEs, and BLG+Lac+DMC / PHL / (DMC+PHL) were acquired using a FIB-SEM GX4 scanning electron microscope (Thermo Fisher Scientific, USA). Samples were mounted on conductive plates and sputtered with gold, and imaging was performed under low vacuum conditions with an accelerating voltage of 10 kV.

[0044] 4.2 SDS-PAGE Study

[0045] SDS-PAGE analysis was performed using a 5% stacking gel and a 12% separating gel. BLG, AGEs, and BLG+Lac+DMC / PHL / (DMC+PHL) sample solutions (2 mg / mL) were mixed with 5... Mix the protein sample loading buffer, boil for 5 min, and centrifuge at 4000 rpm for 5 min. Add 10 μL of the buffer to each well. The supernatant was electrophoresed at 80 V for 30 min, followed by electrophoresis at 120 V for 1.5 h. After electrophoresis, the samples were stained and destained with Coomassie Brilliant Blue R250 and a mixture containing 10% acetic acid and 45% methanol, respectively.

[0046] 4.3. Fluorescence Study of Thionyl T (ThT)

[0047] The inhibitory effect of AGEs on amyloid cross-β structure formation and the individual or synergistic inhibitory effects of DMC and PHL on it were determined using the ThT fluorescence method. BLG, AGEs, or BLG+Lac+DMC / PHL / (DMC+PHL) lyophilized sample solutions (20 μL) were used. M) and ThT(40 Mix with M) and incubate at 25°C for 1 h. At 450–600 nm ( ex The fluorescence spectrum was recorded within the range of 440 nm, and the inhibition rate of ThT fluorescence was calculated using the following formula.

[0048] Equation 4; in F g , F t and F n The ThT fluorescence intensities of AGEs, BLG+Lac+DMC / PHL / (DMC+PHL), and BLG at 485 nm are respectively.

[0049] 5. Research on the mechanisms by which CUR, DMC, and THC inhibit AGEs

[0050] 5.1 Fluorescence Spectroscopic Analysis of the BLG / AGEs+DMC / PHL System

[0051] The fluorescence properties of the BLG / AGEs+DMC / PHL system were evaluated using an F-7000 fluorescence spectrophotometer. The molar concentrations of BLG and AGEs were fixed at 4. The molar concentrations of M, DMC, and PHL were 0-48 μM and 0-64 μM, respectively. The interactions of DMC and PHL with BLG occurred at 25, 31, and 37 °C, while the interactions with AGEs occurred only at 25 °C. After incubation for 30 min, fluorescence spectra were recorded in the 300-450 nm range. ex =280 nm). Background interference and internal filtering effects were corrected before fluorescence intensity data processing.

[0052] 5.2 Ternary Fluorescence Spectroscopy Study

[0053] Fluorescence spectroscopy measurements were performed on (BLG+PHL)+DMC and (BLG+DMC)+PHL to investigate the effects of PHL and DMC on their interaction with BLG. First, a mixed solution of BLG (4 μM) with either PHL or DMC was prepared. Then, different concentrations of DMC and PHL were mixed with the BLG+PHL and BLG+DMC solutions, respectively. The concentrations of DMC and PHL, and other experimental conditions, were the same as for the binary system.

[0054] 5.3 Molecular docking

[0055] Molecular docking analysis of DMC or PHL with BLG was performed using AutoDock Vina software. BLG (ID: 1B0O) was obtained from the RCSB protein database, and the structures of DMC and PHL were obtained from Automated Topologu Builder. The grid size and spacing were 126 Å × 126 Å × 126 Å and 0.375 Å, respectively, covering all active sites. The docking complex with the lowest binding energy was visualized and analyzed using Discovery Studio 2019 software.

[0056] 5.4 Free radical scavenging experiment

[0057] use The synergistic antioxidant capacity of DMC and PHL was evaluated using a free radical scavenging method. ABTS (2.5 mM) and AAPH (1 mM) were mixed and reacted at 68 °C for 40 min in the dark to generate… The solution was cooled to room temperature in the dark and diluted to an absorbance of approximately 0.76 at 734 nm. 2.94 mL of ABTS was taken. + The solution was mixed with 60 μL of DMC or PHL (5-10 μM) solution and incubated at 25°C for 30 min. The experimental clearance rate (ESC) was calculated using Equation 5: Formula 5.

[0058] In the formula A and A 0 represents adding or not adding DMC or PHL, respectively. The absorbance of the solution at 734 nm. The synergistic experiment of DMC and PHL was designed using Design Expert software. The common theoretical clearance and SE of DMC and PHL were calculated using the following Equations 6 and 7: Formula 6; in, ESC 1 and ESC 2 represents the clearance rates of DMC and PHL, respectively.

[0059] Formula 7.

[0060] 6. Cell damage research

[0061] 6.1 ROS Detection

[0062] Intracellular ROS levels were detected using the DCFH-DA fluorescent probe method. Ea.hy926 cells were cultured at 1 × 10⁻⁶ cells per well. 5 Cells were seeded at a density of [number] cells per well in 24-well plates and cultured at 37°C for 24 h. The DMEM medium was then discarded, and all wells were washed with PBS. AGEs (200 [units]) were then used to [determine the cell count]. g / mL), BLG+Lac+DMC / PHL / (DMC+PHL) lyophilized sample (200 g / mL), BLG+Lac+DMC / PHL / (DMC+PHL) g / mL), AGEs (200 g / mL) g / mL) + DMC / PHL / (DMC+PHL)(20 After treating Ea.hy926 cells with M) for 12 h, they were incubated with 10 μM DCFH-DA for 30 min, followed by washing three times with PBS to remove free probes. Fluorescence images were acquired using an inverted fluorescence microscope (IX 73, Olympus, Tokyo, Japan).

[0063] To further quantify intracellular ROS levels, a ROS quantification experiment was performed. Cells were seeded in 96-well plates (5... 10 3 Cells were cultured in wells for 24 h, then pretreated with the above sample solution for another 24 h. After removing the culture medium, the cells were treated with DCFH-DA (10... Cells were incubated in the dark (M, 30 min) and fluorescence intensity was measured using a microplate reader at an excitation wavelength of 485 nm and an emission wavelength of 527 nm. Cell numbers were subsequently corrected using the MTT assay. Cells were washed with PBS after each step of the experiment.

[0064] 6.2 MTT Study

[0065] The MTT assay was used to investigate the cytotoxicity of AGEs on Ea.hy926 cells and the inhibitory effects of DMC and PHL, alone or in synergy, on cytotoxicity. 3 × 10⁶ cells were used per well. 3 Cells were seeded at a density of [number] cells per well in 96-well plates and incubated at 37°C with 5% CO2 for 24 h to allow cell adhesion. AGEs (200 [units]) were selected. g / mL), BLG+Lac+DMC / PHL / (DMC+PHL) lyophilized sample solution (200 g / mL), BLG+Lac+DMC / PHL / (DMC+PHL) g / mL), AGEs (200 g / mL) g / mL) + DMC / PHL / (DMC+PHL)(20 Cells were treated with MTT for 48 h, with 6 replicates per group. After treatment, 20 μL of MTT solution (5 mg / mL) was added to each well, and the cells were incubated in the dark for 4 h. The supernatant was discarded, and 150 μL of DMSO was added to each well to dissolve the blue-purple formazan crystals. The absorbance of each well was measured at 570 nm using a microplate reader.

[0066] 6.3 Apoptosis Research

[0067] Apoptosis was detected using Annexin V-FITC / PI double staining. Ea.hy926 cells were seeded in 6-well plates (15 μL each). 10 4 Cells were cultured in one well for 12 h. Cells were then treated with the above sample for 24 h; cells cultured in the same medium served as a control group. Cells were collected, stained with Annexin V-FITC and PI, and then analyzed by flow cytometry.

[0068] 7. Statistical Analysis

[0069] Each experiment was repeated three times, and one-way ANOVA was performed using SPSS 19.0 software. The measured values... p A value less than 0.05 is considered significant.

[0070] II. Results and Discussion

[0071] 1. Analysis of the synergistic inhibition of AGEs by DMC and PHL

[0072] 1.1 Cooperative inhibition ability

[0073] like Figure 1 As shown in Figures A and B, the inhibitory effects of DMC and PHL on AGEs are concentration-dependent, indicating that DMC and PHL can inhibit AGE formation. The IC50 values ​​for DMC and PHL are... 50 183 respectively M and 47 M indicates that DMC has a stronger ability to inhibit AGEs than PHL, which may be related to their binding strength with BLG and their antioxidant mechanisms, and warrants further investigation.

[0074] To investigate the synergistic inhibitory effect of DMC and PHL on AGEs, the concentration of one polyphenol was kept constant, and the effect of changing the concentration of the other polyphenol on the synergistic coefficient was examined. The concentrations of DMC and PHL were kept constant at 40 mg / L. M and 150 M, as the concentration of PHL increases from 100 to 225 M, DMC concentration increased from 25 to 60 M, the synergy coefficient is greater than 1 ( Figure 1 The decreasing trend of C and D indicates that both DMC and PHL have a synergistic ability to inhibit AGEs formation within a certain concentration range. This may be related to mechanisms such as competition for glycosylation sites and synergistic antioxidant activity. However, with increasing concentrations of DMC and PHL, although the degree of inhibition increases, the synergistic coefficient decreases. This may be due to the increasing concentration leading to molecular crowding, which interferes with their interaction with BLG, hindering the synergistic effect and causing the synergistic coefficient to continuously decrease.

[0075] 1.2 Carbonyl and thiol group content

[0076] The carbonyl and thiol content of proteins is a recognized indicator of protein oxidation. During Lac-induced BLG glycosylation, a large number of free radicals attack the amino acid side chains, oxidizing them to carbonyl groups. Simultaneously, glycosylation alters the tertiary structure of BLG, exposing thiol groups originally embedded within the protein to the molecular surface, making them more susceptible to free radical oxidation to disulfide bonds, thus decreasing the free thiol content. As shown in Table 1, treatment with DMC and PHL alone or in combination resulted in decreased and increased carbonyl and thiol contents, respectively, indicating that both DMC and PHL effectively inhibit glycosylation-induced BLG oxidation. The carbonyl contents of the DMC-PHL and DMC-PHL combined systems were 20.32±0.25, 18.65±0.22, and 12.71±0.14 µmol / g, respectively, while the thiol contents were -25.32±0.41, -25.96±0.45, and -23.27±0.32 µmol / g, respectively. The order of inhibition of carbonyl and thiol changes is DMC + PHL > PHL > DMC. PHL's ability to inhibit AGEs formation is lower than DMC, but its ability to inhibit carbonyl increase and thiol decrease is stronger, possibly due to its stronger direct antioxidant capacity, enabling it to more effectively scavenge reactive oxygen species and thus inhibit BLG oxidation. Furthermore, the combination of DMC and PHL has a better effect on inhibiting carbonyl increase and thiol decrease than a single polyphenol, possibly due to their synergistic effect and superior antioxidant capacity.

[0077] Table 1. Carbonyl, mercapto, and relative amino content of BLG, AGEs, BLG+Lac+DMC, BLG+Lac+PHL, and BLG+Lac+PHL+DMC. D h and Zeta potential value

[0078] 1.3 Amino content

[0079] Amino groups can serve as an indicator of the degree of protein glycosylation. The OPA method was used to study the ability of DMC and PHL, alone or in combination, to protect amino groups. Figure 2As shown in Table 1, glycosylation reduced the free amino content to 43.2% of the natural BLG level, indicating that Lac induces glycosylation in BLG. After treatment with DMC (BLG+Lac+DMC) or PHL (BLG+Lac+PHL) in the glycosylation system, the relative amino content recovered by approximately 22.6% and 18.7%, respectively. This result indicates that both DMC and PHL effectively protect the free amino groups of BLG, reducing their covalent binding with Lac in the early stages of glycosylation. DMC's ability to protect amino groups is stronger than that of PHL, consistent with its ability to inhibit AGEs, which may be related to the difference in binding strength between DMC or PHL and BLG. The system treated with both PHL and DMC (BLG+Lac+PHL+DMC) showed an increase in relative amino content of approximately 42.6%, greater than the sum of the recovery abilities of the individual components, exhibiting a synergistic protective effect. This suggests that the two have a synergistic effect in inhibiting the early stages of the Maillard reaction.

[0080] 2. Conformational Change Analysis

[0081] 2.1 CD spectral analysis

[0082] CD spectroscopy is a commonly used method for elucidating the secondary structure of proteins. After Lac induction, the peak intensity of the BLG band significantly decreased, indicating that glycosylation altered the secondary structure of BLG. However, under the influence of DMC and PHL, alone or in combination, the CD spectral intensity recovered to some extent, becoming closer to that of the pure protein. Figure 3 A). This indicates that DMC and PHL can prevent the effects of glycosylation on the secondary structure of BLG. The secondary structure composition is as follows: Figure 3 As shown in Figure C, the α-helix, β-sheet, β-turn, and random coil contents of natural BLG are 12.6%, 36.1%, 21.6%, and 29.7%, respectively. After glycosylation, the composition of BLG... -The content of helical and β-sheet structures decreased by 4.5% and increased by 10.2%, respectively. After the addition of DMC or PHL, The α-helix and β-sheet increased by 2.0% and 1.5%, respectively, while the β-sheet decreased by 4.9% and 4.0%, respectively. This indicates that DMC and PHL can stabilize the structure of BLG and protect it from glycosylation. The stronger protective effect of DMC on the secondary structure of BLG compared to PHL is likely due to DMC's stronger binding affinity to BLG. In the co-existence of DMC and PHL, the α-helix and β-sheet increased and decreased by 2.6% and 6.5%, respectively, which is superior to the single inhibitor system, indicating that the co-existence of both can better stabilize the structure of BLG.

[0083] 2.2 DLS Research

[0084] Particle size distribution map and Zeta potential values ​​are as follows Figure 3 B and D, and as shown in Table 1, natural BLGD h The zeta potentials were 4.84 ± 0.03 nm and -18.82 ± 0.40 mV, respectively, consistent with literature values. The glycosylation of BLG... D h As the zeta potential increases, it becomes more negative. This is due to the binding of Lac and the introduction of more negative charges. In the presence of DMC or PHL, D h Both the molecular weight and potential of BLG were restored, becoming closer to that of natural BLG. This further demonstrates that they can stabilize the structure of BLG and protect it from glycosylation-induced modifications. The stronger protective ability of DMC may stem from its stronger affinity for BLG. When DMC and PHL coexist, D h The better potential recovery effect indicates that the combination of DMC and PHL can better inhibit glycosylation-induced BLG structural changes.

[0085] 3. Cluster analysis

[0086] 3.1 SEM Analysis

[0087] The effects of DMC and PHL on glycosylation-induced BLG aggregation were investigated by directly observing the morphology of BLG using SEM. Figure 4 As shown in Figures A through E, natural BLG is a smooth, spherical structure. Glycosylation leads to the formation of numerous aggregates, indicating that glycosylation induces BLG aggregation. In the presence of DMC or PHL, the aggregates become looser, suggesting that DMC and PHL effectively inhibit BLG aggregation induced by glycosylation. The degree of dispersion is not significantly different in the presence of DMC and PHL, possibly due to the presence of 40... M's DMC with 150 The ability of DMC and PHL to inhibit AGEs production is not significantly different, making it difficult to distinguish them visually using SEM. When DMC and PHL coexist, the aggregates are more loosely distributed and dispersed into smooth spheres with shapes similar to natural BLG, indicating that the combined use of the two can exert a significant synergistic anti-aggregation effect.

[0088] 3.2 SDS-PAGE Analysis

[0089] like Figure 5As shown in Figure A, the bands at 17 kD and 34 kD are characteristic bands of BLG monomers and dimers, respectively. The AGEs band lightens in color at 17 kD but deepens at 34 kD, indicating that glycosylation promotes BLG cross-linking and aggregate formation. After inhibition with both polyphenols, the color at 17 kD deepens and the color at 34 kD lightens, more closely resembling pure protein. The color recovery after DMC inhibition is more pronounced than after PHL inhibition, indicating that DMC has a stronger ability to inhibit BLG aggregation than PHL, consistent with its ability to inhibit AGEs. The combined polyphenols show a more significant color recovery, suggesting that the combined polyphenols have a stronger effect than a single polyphenol.

[0090] 3.3 ThT fluorescence analysis

[0091] like Figure 5 As shown in Figure B, the fluorescence intensity of AGEs significantly increased compared to native BLG after binding to ThT. This indicates that glycosylation leads to protein aggregation and the formation of amyloid cross-beta structures. The fluorescence intensity significantly decreased when DMC and PHL were present alone or together, indicating that DMC and PHL can inhibit the formation of amyloid structures. Furthermore, at the same concentration, the inhibition rates of DMC and PHL were 34.7% and 28.0%, respectively, consistent with their ability to inhibit AGEs formation. The inhibition rate of DMC and PHL in combination was 67.4%, greater than the sum of their individual inhibition rates, suggesting that DMC and PHL can synergistically inhibit AGEs-induced BLG fibrosis.

[0092] 4. Research on inhibition mechanisms

[0093] 4.1 Fluorescence Quenching Analysis

[0094] Occupying protein glycosylation sites is one of the main mechanisms by which polyphenols inhibit glycosylation. To investigate this mechanism, this study used fluorescence spectroscopy to examine the binding of DMC and PHL to BLG / AGEs. This method is widely used to study ligand-biomacromolecule interactions. Figure 6As shown in Figures A through D, at an excitation wavelength of 280 nm, the maximum fluorescence emission wavelength of BLG is 337 nm, while the maximum emission wavelength of AGEs red-shifts to 344 nm, indicating that glycosylation causes structural changes in BLG. Previous studies in our group found that fructose-induced maximum emission wavelength of HSA shifted from 338 nm to 314 nm. With the addition of DMC and PHL, the fluorescence signals of both BLG and AGEs gradually decreased. When DMC binds to BLG and AGEs, the maximum emission wavelengths show significant blue shifts of 6.2 nm and 2.4 nm, respectively. In the presence of PHL, BLG and AGEs exhibit slight red shifts of only 1 nm and 0.8 nm, respectively. This indicates that the binding of DMC to BLG / AGEs places the fluorophores of BLG and AGEs in a more hydrophobic environment, while the binding of PHL places them in a more hydrophilic environment. Furthermore, at the same concentration, DMC has a stronger quenching effect on BLG than PHL, indicating that DMC has a stronger binding affinity to BLG than PHL.

[0095] To elucidate the interaction between DMC and PHL with BLG / AGEs and their relationship with glycosylation inhibition, this study first employed fluorescence spectroscopy to analyze the quenching mechanism. The quenching rate constant ( k q Calculated using the following formula: Formula 8; in, F 0 and F These represent the fluorescence intensities of the BLG / AGEs and BLG / AGEs+DMC / PHL systems after background correction and internal filtration compensation, respectively; Q ]0 represents the molar concentration of the two polyphenols; τ0 represents the fluorescence lifetime of BLG / AGEs.

[0096] Based on the linear Stern-Volmer plot ( Figure 6 The quenching rate constant calculated from the illustration ( k q (Table 2) shows a negative correlation with temperature, and its value is greater than the diffusion-controlled limiting quenching constant (2 × 10⁻⁶). 10 L·mol -1 ·s -1 This indicates that the quenching mechanism of DMC and PHL on BLG / AGEs is static quenching.

[0097] For static quenching mechanisms, combined with constants ( K a ) and number of binding sites ( n It can be calculated using the following formula: Equation 9; in,[Q The numbers [] represent the free concentrations of DMC and PHL. As shown in Table 2, n A value close to 1 indicates that DMC and PHL share a binding site on BLG and AGEs. Comparison at the same temperature... K a It is known that the binding strength between DMC and BLG is stronger than that between DMC and PHL. This may be because the two benzene rings of DMC are connected by an enone to form a rigid planar conjugated system, making its binding with BLG more stable. Secondly, even though PHL has a higher hydroxyl content than DMC, the methoxy group of DMC is a strong electron-donating group, which can enhance the electron cloud density of its benzene ring, making the binding more stable. Furthermore, within the temperature range of 25℃-37℃, as the temperature increases... K a The decrease in temperature indicates that higher temperatures are unfavorable for binding. Furthermore, the affinity of PDMC and HL for AGEs is significantly lower than their affinity for BLG. This suggests that glycosylation hinders the binding of DMC and PHL to BLG, meaning that DMC or PHL may inhibit Lac-induced BLG glycosylation by occupying glycosylation sites.

[0098] The driving force behind the binding process of DMC and PHL with BLG can be determined by enthalpy change ( H ) and entropy change (Δ S °) is determined, and the calculation equation is as follows: Formula 10; As shown in Table 2, negative Δ G The ° value proves that DMC and PHL spontaneously bind to BLG. The negative Δ H The ° value indicates that van der Waals forces, hydrogen bonds, and electrostatic interactions affect Δ H The negative contribution of Δ is greater than the positive contribution of hydrophobic interactions. Positive Δ S The ° value indicates that the entropy increase from the release of water molecules during the binding process exceeds the entropy decrease from the formation of the complex. The Δ value for the BLG+DMC system... H The ΔHo is more negative than the BLG+PHL system, possibly due to the stronger binding force between DMC and BLG, resulting in greater heat release. The ΔHo of the BLG+DMC system is also higher. S The lower ° value compared to BLG+PHL may be due to the stronger conformational constraint imposed on the complex by the strong binding between them.

[0099] 4.2 Ternary fluorescence analysis

[0100] To investigate the interactions between DMC, PHL, and BLG, this study conducted fluorescence experiments on the BLG+DMC+PHL ternary system. Figure 7As shown, in the ternary system, the fluorescence intensity of BLG+PHL / DMC decreases with increasing DMC / PHL concentration. The fluorescence intensity of the BLG+DMC+PHL ternary system was obtained using Matlab R2016a software based on a competition model fitting. K a and n The thermodynamic parameters are then calculated using formula 2-10.

[0101] At the same temperature, the binding strength of DMC and PHL to BLG in the ternary system is lower than that in the corresponding binary system. At 25℃, the presence of PHL reduces the binding strength of BLG+DMC. K a Decreased by 60.8% (from 4.03 ± 0.11 × 10⁻⁶) 5 It decreased to 1.58 ± 0.11 × 10⁻⁶. 5 The presence of DMC makes BLG+PHL K a decreased by 70.0% (from 1.36 ± 0.09 × 10⁻⁶). 5 It decreased to 0.40±0.02×10 5 The more pronounced decrease in the PHL binding constant is likely because the stronger affinity of DMC for BLG hinders PHL binding. This indicates that both polyphenols bind to the same site on BLG.

[0102] DMC and PHL compete to bind to the glycosylation site of BLG. The simultaneous binding of the two polyphenols may lead to greater steric hindrance, making it more difficult for lysine residues to be exposed and react with Lac to produce AGEs, thereby achieving a synergistic effect of inhibiting AGEs.

[0103] In a ternary system, Δ H °<0 and Δ S °>0 indicates that the binding mode and driving force between DMC or PHL and BLG may remain unchanged in the presence of PHL or DMC. In the (BLG+PHL)+DMC system, Δ H The absolute value of ° is larger than that of Δ S The smaller ° value may be due to the fact that the binding strength between DMC and BLG is greater in the presence of PHL than that between PHL and BLG in the presence of DMC. This results in more heat release in the (BLG+PHL)+DMC system and imposes stronger conformational constraints on the complex.

[0104] Table 2 Quenching rate constants and thermodynamic parameters of the interaction between DMC and / or PHL and BLG at different temperatures

[0105] 4.3 Molecular docking research

[0106] The optimal binding site was selected based on the lowest binding energy. The lowest binding energies of DMC and PHL to BLG were -34.93 and -30.26 kJ / mol, respectively, further indicating that DMC binds more strongly to BLG. Figure 8 As shown in Figures A-B, DMC and PHL share numerous identical amino acids (LYS 60, LYS 69, LEU 39, PRO 38, etc.) at their docking sites on BLG, indicating that they bind to the same sites on BLG, thus validating the conclusion of ternary fluorescence. Furthermore, DMC binds to LYS 60 and LYS 69 on BLG via hydrogen bonds and van der Waals forces, respectively, while PHL binds to LYS 60 and LYS 69 via van der Waals forces. LYS 60 and LYS 69 are common glycosylation sites for Lac-induced BLG glycosylation, suggesting that DMC and PHL can inhibit AGEs production by occupying these glycosylation sites on BLG.

[0107] To further investigate the mechanism by which DMC and PHL synergistically inhibit AGEs production, ternary molecular docking was performed. In the presence of DMC, the lowest binding energies of PHL and BLG were -26.76 kJ / mol, and in the presence of PHL, the lowest binding energies of DMC and BLG were -29.64 kJ / mol, consistent with the findings of ternary fluorescence. Figure 9 As shown in A~B, both DMC and PHL are bound in the hydrophobic cavity of BLG. When either PHL or DMC is present, DMC and PHL are bound at their adjacent positions.

[0108] The results of ternary molecular docking indicate that DMC and PHL have greater steric hindrance when they coexist, which further confirms the hypothesis of the ternary fluorescent system. The greater steric hindrance makes it more difficult for lysine residues to be exposed and react with Lac to produce AGEs, thereby achieving a synergistic inhibition of AGEs.

[0109] 4.4 Antioxidant Capacity Study

[0110] via ABTS + Free radical scavenging experiments evaluated the synergistic antioxidant capacity of DMC and PHL. Figure 10 As shown in Figure A, both DMC and PHL can effectively scavenge free radicals in a concentration-dependent manner. 50 The values ​​were 7.52 ± 0.05. M and 5.58±0.07 The value of M indicates that PHL has a greater antioxidant capacity than DMC. PHL's stronger antioxidant properties may be due to the presence of more hydroxyl groups in its molecular structure. Phenolic hydroxyl groups in phenolic compounds can donate hydrogen atoms to free radicals, transforming themselves into relatively stable phenoxy radicals, thus quenching free radicals. While PHL has a stronger antioxidant capacity than DMC, its ability to inhibit AGEs is weaker. This may be because AGE formation involves multiple stages, and different polyphenols may play different roles at each stage.

[0111] Thirteen experimental schemes were constructed using Design-Expert software. The design parameters and calculation results are shown in Table 3. (Three-dimensional response surface plot) Figure 10 B) shows that when the concentrations of DMC and PHL are 7... M and 6 At time M, the optimal SE value was 1.27, indicating that DMC and PHL have synergistic antioxidant capabilities, suggesting that DMC and PHL may achieve synergistic inhibition of AGEs through synergistic antioxidant effects. The synergistic antioxidant capability of DMC and PHL may be due to the fact that the 2,6-dihydroxyacetophenone structure of PHL is a potent hydrogen donor, capable of rapidly quenching free radicals and generating phenoxy radicals, while DMC, due to its strong electron delocalization ability, can donate electrons or hydrogen atoms, allowing the regeneration of oxidized PHL free radical intermediates, thus achieving synergistic antioxidant capabilities.

[0112] Table 3. Box-Behnken experimental design and results for synergistic optimization of DMC and PHL.

[0113] 5. Cell damage analysis

[0114] 5.1 ROS Analysis

[0115] ROS play a crucial role in the development and progression of CDV, therefore it is necessary to investigate the scavenging effects of DMC and PHL, alone or in synergy, on AGEs-induced ROS. (Image from fluorescence microscopy) Figure 11 As shown in Figures A through H, the fluorescence intensity of cells treated with AGEs was significantly enhanced, indicating the production of more ROS. This may be because AGEs bind to the AGEs receptor RAGE on the cell surface, activating NADPH oxidase and leading to a large amount of intracellular ROS production. The fluorescence intensity of cells treated with BLG+Lac+DMC / PHL was significantly reduced, and the fluorescence intensity of cells treated with both AGEs and DMC / PHL was also reduced. This suggests that the two polyphenols can both reduce ROS production by inhibiting AGEs and inhibit AGEs-induced ROS production in cells.

[0116] To further compare the ability of the two polyphenols to inhibit ROS individually or together, a quantitative ROS experiment was performed. Figure 12 As shown in Figure A, the fluorescence intensity of cells treated with AGEs was 2.32 times that of the control group. The fluorescence intensities of cells treated with DMC, PHL, and DMC+PHL (which inhibited AGEs) were 1.54, 1.68, and 1.34 times that of the control group, respectively, indicating that inhibiting AGEs production can alleviate cell damage. Cells treated with DMC, PHL, and DMC+PHL in combination with AGEs showed a 27%, 46%, and 81% reduction in fluorescence intensity compared to cells treated with AGEs alone, respectively, indicating that DMC and PHL can effectively inhibit AGEs-induced cellular oxidative stress and have a synergistic effect. DMC may reduce ROS production by inhibiting NADPH oxidase, while PHL may alleviate oxidative stress by activating the Nrf2 signaling pathway. Both may synergistically inhibit ROS production through different mechanisms. These studies suggest that DMC and PHL can effectively prevent the occurrence and development of CDV.

[0117] 5.2 MTT Analysis

[0118] Depend on Figure 12 As shown in Figure B, BLG treatment had virtually no effect on the viability of Ea.hy926 cells, but cell viability decreased significantly after treatment with glycosylated BLG. This is likely due to the large amount of ROS induced by AGEs, leading to decreased cell viability. Cells treated with AGEs inhibited by DMC or PHL alone or in combination showed some recovery in cell viability. This is likely because DMC and PHL inhibited AGEs production, reducing ROS production, alleviating cellular inflammation, and decreasing apoptosis, thereby improving cell survival. Cells treated with DMC, PHL, and DMC+PHL in combination with AGEs showed increased cell viability by 10.0%, 14.7%, and 36.7%, respectively, compared to cells treated with AGEs alone, indicating that DMC and PHL can inhibit AGEs-induced cytotoxicity and have a synergistic effect. This may be due to the strong antioxidant capacity of DMC and PHL, which can directly eliminate AGEs-induced ROS and thus reduce cytotoxicity. PHL's stronger protective ability may be due to its stronger antioxidant capacity, which can eliminate more ROS.

[0119] 5.3 Apoptosis Analysis

[0120] Apoptosis is closely related to CDV (Chronic Disease Vulnerability) and is a core pathological step in the disease's development. Apoptosis was detected in Ea.hy926 cells after treatment with samples for 24 hours. Figure 13As shown in A and B, compared with BLG, the proportion of apoptosis significantly increased after AGEs induction, with the ratio of late apoptosis + early apoptosis reaching 53.4% ​​(Q1: mechanically damaged cells, Q2: late apoptotic cells, Q3: early apoptotic cells, Q4: normal cells). This may be because the excessive ROS induced by AGEs disrupts mitochondrial function, directly damaging DNA, proteins, and lipids, triggering apoptosis signaling pathways, and leading to apoptosis. Figure 13 CE showed that the apoptosis rate of AGEs inhibited by DMC and PHL was significantly reduced, especially the combined inhibition had a more significant effect, indicating that polyphenols can effectively reduce cell apoptosis by inhibiting the generation of AGEs.

[0121] Depend on Figure 13 FH analysis revealed that DMC and PHL can also directly protect cells from AGEs-induced apoptosis. At the same concentration, DMC inhibited apoptosis by 16.4%, while PHL inhibited it by 35.2%. PHL's stronger anti-apoptotic ability may be due to its stronger antioxidant capacity, which better scavenges AGEs-induced ROS. The combined inhibition rate of both was 68.6%, indicating that the co-existence of DMC and PHL more effectively inhibits AGEs-induced apoptosis. These results suggest that DMC and PHL exhibit a synergistic effect in cell models, hinting at their potential therapeutic value in the intervention of CDV.

[0122] As shown in the above examples, this invention provides the application of demethoxycurcumin and phlorizin in the preparation of drugs that inhibit β-lactoglobulin glycosylation, and systematically explores the synergistic inhibitory effects and mechanisms of demethoxycurcumin and phlorizin on AGEs. Fluorescence spectroscopy and carbonyl / thiol / amino group assays confirmed that demethoxycurcumin and phlorizin can synergistically inhibit β-lactoglobulin glycosylation at appropriate concentrations. Circular dichroism spectroscopy and other experiments showed that the combined use of the two can better alleviate changes in protein secondary structure and aggregation. They competitively bind to the same glycosylation site, synergistically resist oxidation, thereby inhibiting AGEs formation, and synergistically reducing oxidative stress, cell damage, and apoptosis at the cellular level. Therefore, the combined application of these two has potential therapeutic value in intervening in diabetic complications.

[0123] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A functional composition, characterized in that, It includes the following components: demethoxycurcumin and phloretin.

2. The use of the composition according to claim 1 in the preparation of an anti-glycation drug.

3. The application according to claim 2, characterized in that, The concentration of demethoxycurcumin in the anti-glycation drug is 30-50 μM, the concentration of phloretin in the anti-glycation drug is 140-160 μM, and the anti-glycation drug is a drug that inhibits the formation of advanced glycation end products.

4. The use of the composition according to claim 1 in the preparation of antioxidant drugs.

5. The application according to claim 4, characterized in that, The concentration of demethoxycurcumin in the antioxidant drug is 5-9 μM, and the concentration of phloretin in the antioxidant drug is 4-8 μM.

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