Application of cyanidin-3-O-glucose or combination of cyanidin-3-O-glucose and synephrine to removal of 4-hydroxy-2-nonenal

Abstract

The invention discloses an application of cyanidin-3-O-glucose or a combination of cyanidin-3-O-glucose and synephrine in removing 4-hydroxy-2-nonenal, and discloses a new application of C3G for the first time, namely the C3G can effectively capture 4-hydroxy-2-nonenal (4-HNE) to generate an adduct product so as to control the content of the adduct product. The condition that the chitosan is combined with nucleophilic groups in biomacromolecules to form a cross-linked product to hinder normal running of body functions is avoided. In addition, when the cyanidin-3-O-glucose and the synephrine are in binary combination, the synephrine can be used as a catalyst to promote the cyanidin-3-O-glucose to remove 4-HNE. The cyanidin-3-O-glucose and the synephrine are combined to quickly and effectively capture 4-HNE generated in an environment, in a living body or in inhibition of food production and storage, so that the safety of the environment and food is improved, and the harm to a human body after the 4-HNE is taken into the body is blocked.

Description

Technical Field

[0001] The invention belongs to the application field of anthocyanins and alkaloids, and specifically relates to the application of cyanidin-3-O-glucose or cyanidin-3-O-glucose combined with synephrine for removing 4-hydroxy-2-nonenal. Background Art

[0002] 4-Hydroxy-2-nonenal (4-HNE) is a highly toxic α,β-unsaturated carbonyl compound that is closely related to the oxidative decomposition of unsaturated fatty acids and is one of the most representative end products of lipid peroxidation. 4-HNE has high reactivity to DNA and proteins and is widely accepted as a biomarker of oxidative stress in the field of biological research.

[0003] As a highly active α,β-unsaturated aldehyde, 4-HNE can quickly enter physiological tissues. And because of its strong electrophilicity, it undergoes electrophilic addition reactions with cellular nucleophilic substances such as proteins, DNA, RNA and other biological macromolecules, resulting in changes in the structure of biological macromolecules, which in turn induces physiological abnormalities and causes cancer, Alzheimer's disease, Parkinson's disease, rheumatoid arthritis and other diseases. Studies have found that the reactivity of 4-HNE with proteins is higher than that of DNA. After being oxidized in the body, it immediately binds to proteins. The substances formed by Michael addition of 4-HNE with cysteine, histidine and lysine residues in proteins are relatively stable in nature. After accumulating in the body, they show certain toxic effects. Secondly, 4-HNE will promote oxidative stress in the body, destroy the original redox balance in the body, and cause changes in the body's physiological state.

[0004] 4-HNE is produced in two ways: endogenous and exogenous. During food processing, 4-HNE is mainly derived from the peroxidation of ω-6 polyunsaturated fatty acids, including linoleic acid (LA), γ-linolenic acid (GLA) and arachidonic acid (AA). The process of lipid oxidation to produce 4-HNE can be divided into two stages: fatty acid oxidation to lipid hydroperoxide (LOOH) and LOOH decomposition to 4-HNE. 4-HNE in food is easily absorbed from the intestine into the systemic circulation. The European Food Safety Authority (EFSA) Scientific Committee's recommendation on the toxicological concern threshold (TTC) of 4-HNE indicates that the TTC of 4-HNE is 1.5 μg / kg body weight / day. At present, the content of 4-HNE in general frying vegetable oil is: 1.6-4.8 μg / g, the content of 4-HEN in baked and fried meat products is: 0.36-2 μg / g, the content of 4-HNE in infant formula is as high as 2.85 μg / g, the content of 4-HNE in tea and coffee is: 0.6-1.08 μg / mL, and the content of 4-HNE in dark beer is 0.8-2.2 μg / mL. If the proportion of high-fat foods in the diet is too high, such as bacon, French fries and fried chicken, there is a risk of overexposure to 4-HNE. In the body, 4-HNE is produced by peroxidation of liver microsomal lipids and biomembrane lipids caused by free radicals or chemicals. In addition, 4-HNE can exist in the water environment, causing environmental pollution.

[0005] At present, there are relatively few reports on exogenous additives that inhibit 4-HNE. Except for phloretin, EGCG, quercetin, elderberry plant extracts, and wheat-bound polyphenols, other food-borne flavonoid inhibitors have not been reported. Therefore, continuing to find natural, safe, and efficient substances to capture 4-HNE to reduce the level of 4-HNE in food or in the body and exploring its mechanism is one of the necessary means to prevent environmental pollution and prevent chronic diseases, which has important practical significance and theoretical value.

[0006] Cyanidin-3-O-glucoside (C3G) is an anthocyanin, belonging to the flavonoids. It is widely found in colored grains, fruits and vegetables such as bayberry, blackberry, purple cabbage and black rice. It has good water solubility and good physiological activities, such as anti-oxidation, anti-inflammatory and antibacterial. Synephrine (SYN) is an alkaloid, also known as hydroxyfrine and deoxyepinephrine. It is mainly found in the flowers, fruits and peels of plants of the genus Citrus in the Rutaceae family, including Citrus aurantium, Citrus aurantium, various citrus fruits, lemons, grapefruits, tangerine peels, and daisy flowers. At the same time, synephrine has also been detected in extracts of other plants such as Evodia rutaecarpa, malt and cactus. It has the effects of raising blood pressure, resisting shock, promoting metabolism, burning fat, anti-inflammatory and anti-cancer. In addition, it also has good therapeutic effects on digestive problems and depression. At present, no research has been found on cyanidin-3-O-glucoside and synephrine in inhibiting 4-hydroxy-2-nonenal. Summary of the invention

[0007] Purpose of the invention: In view of the problems existing in the prior art, the present invention provides the use of cyanidin-3-O-glucose or synephrine to catalyze cyanidin-3-O-glucose to remove 4-hydroxy-2-nonenal. In the present invention, cyanidin-3-O-glucose has a good effect of inhibiting 4-hydroxy-2-nonenal, and when it is used in binary combination with synephrine, only a small amount of synephrine is needed as a catalyst to greatly improve the capture activity of cyanidin-3-O-glucoside, thereby inhibiting 4-hydroxy-2-nonenal. In the present invention, cyanidin-3-O-glucose or synephrine catalyzes cyanidin-3-O-glucose to remove 4-HNE, thereby inhibiting 4-hydroxy-2-nonenal present in a natural environment solution system; cyanidin-3-O-glucoside and synephrine, as an anthocyanin contained in food raw materials such as fruits and vegetables, can inhibit 4-hydroxy-2-nonenal during food processing, reduce the content of 4-hydroxy-2-nonenal in food, and fill the gap of 4-hydroxy-2-nonenal inhibitors in food systems.

[0008] The invention also provides a 4-hydroxy-2-nonenal inhibitor in the environment, food processing or in a living body.

[0009] Technical solution: In order to achieve the above-mentioned purpose, the cyanidin-3-O-glucose or synephrine or cyanidin-3-O-glucose and synephrine described in the present invention are used in the removal or inhibition of 4-hydroxy-2-nonenal.

[0010] The invention discloses an application of cyanidin-3-O-glucose or synephrine or a combination of cyanidin-3-O-glucose and synephrine in the preparation of an inhibitor for inhibiting or removing 4-hydroxy-2-nonenal.

[0011] The structure of cyanidin-3-O-glucoside or synephrine of the present invention is as follows:

[0012]

[0013] Among them, the synephrine catalyzes cyanidin-3-O-glucose in inhibiting or removing 4-hydroxy-2-nonenal.

[0014] Furthermore, the cyanidin-3-O-glucose or synephrine catalyzes cyanidin-3-O-glucose to capture 4-hydroxy-2-nonenal, thereby reducing the content of 4-hydroxy-2-nonenal.

[0015] Among them, the application of cyanidin-3-O-glucose or cyanidin-3-O-glucose combined with synephrine in inhibiting 4-hydroxy-2-nonenal in an environment.

[0016] Among them, the cyanidin-3-O-glucose or the cyanidin-3-O-glucose combined with synephrine is used to inhibit 4-hydroxy-2-nonenal produced in food processing.

[0017] Among them, the cyanidin-3-O-glucose or the cyanidin-3-O-glucose combined with synephrine is used to eliminate 4-hydroxy-2-nonenal in organisms.

[0018] Among them, the cyanidin-3-O-glucose or the combination of cyanidin-3-O-glucose and synephrine is used to inhibit various harmful addition or cross-linking products formed by the reaction of 4-hydroxy-2-nonenal with nucleophilic biological macromolecules in the preparation environment, food processing or 4-hydroxy-2-nonenal inhibitors in organisms.

[0019] The cyanidin-3-O-glucose or cyanidin-3-O-glucose is used in combination with synephrine to capture 4-hydroxy-2-nonenal to form an addition product, thereby reducing the content of 4-hydroxy-2-nonenal.

[0020] The invention discloses an application of cyanidin-3-O-glucose or a combination of cyanidin-3-O-glucose and synephrine to remove 4-HNE in the preparation of a drug for preventing chronic diseases in the human body.

[0021] Wherein, the chronic human diseases include Alzheimer's disease, Parkinson's disease, rheumatoid arthritis or cardiovascular and cerebrovascular diseases.

[0022] The amount of cyanidin-3-O-glucose used is 0.5-8.0 mmol / L, and the amount of synephrine used is 0.25-4.0 mmol / L. The amount of synephrine used is 0-2 times the molar amount of cyanidin-3-O-glucose.

[0023] Preferably, the amount of cyanidin-3-O-glucose used is 0.5-8.0 mmol / L; the amount of synephrine used is 0.25-0.5 times the molar ratio of cyanidin-3-O-glucose, and more preferably 0.5 times.

[0024] The present invention discloses an inhibitor of 4-hydroxy-2-nonenal in the environment, food processing or in a living organism. The inhibitor is a preparation formed by using cyanidin-3-O-glucoside or cyanidin-3-O-glucose and synephrine as the sole or main component and compounding with other substances for use together.

[0025] Among them, the cyanidin-3-O-glucose forms an addition product after capturing 4-HNE, and the reaction mechanism of C3G capturing 4-HNE is Michael addition reaction, that is, after C3G and 4-HNE undergo 1,4-addition reaction under alkaline conditions, the hydroxyl group on the A ring of the C3G structure reacts with the aldehyde group on 4-hydroxy-2-nonenal to form or not form a hemiacetal structure, thereby reducing the HNE content. It effectively fills the gap of 4-hydroxy-2-nonenal inhibitors in the environment, in the food processing system on the market, and in organisms.

[0026] Among them, the cyanidin-3-O-glucoside can play a role in the natural form of mulberry and bayberry, and synephrine can play a role directly in the natural form of tangerine peel and orange.

[0027] The addition product includes an addition product C3G-HNE-1 of cyanidin-3-O-glucose and 4-hydroxy-2-nonenal, and its structure is shown below:

[0028]

[0029] When cyanidin-3-O-glucose and synephrine are used in combination, synephrine as a catalyst greatly improves the capture activity of cyanidin-3-O-glucose, thereby achieving the effect of inhibiting 4-hydroxy-2-nonenal.

[0030] The cyanidin-3-O-glucoside described in the present invention is the main anthocyanin substance in fruits and vegetables, and the synephrine is the main alkaloid substance in the flowers, fruits and peels of plants of the genus Citrus in the Rutaceae family, including Citrus aurantium, Citrus aurantium, lemon, grapefruit, tangerine peel, Citrus grandiflora, various citrus fruits, and other plants such as Evodia rutaecarpa, malt and cactus.

[0031] Although the research on α,β-unsaturated carbonyl compound inhibitors is relatively extensive, there are few inhibitors developed for 4-HEN, and due to factors such as thermal stability and safety, there are not many inhibitors that can be used in food and the human body. The inhibitor developed by the present invention, C3G alone or in combination with SYN, can be used in food and the human body with natural foods such as fruits and vegetables as a medium. The C3G of the present invention itself has a certain inhibitory effect on 4-HNE, and after being combined with SYN, its inhibitory activity can be significantly improved.

[0032] The present invention proposes for the first time that C3G can capture 4-HNE to form an additive product to achieve the effect of removing or inhibiting 4-HNE. Furthermore, the present invention proposes for the first time that SYN can promote or catalyze the activity of C3G in capturing 4-HNE, significantly improving the ability of C3G to remove 4-HNE, while SYN alone cannot inhibit or remove 4-HNE. This is different from the previous mechanism of removing active carbonyl compounds by flavonoids or alkaloids alone or in combination. The present invention experiments prove that after adding a small amount of synephrine, the inhibitory effect of C3G on 4-HNE is greatly improved.

[0033] Beneficial effects: Compared with the prior art, the present invention has the following advantages:

[0034] The present invention proposes for the first time a new application of cyanidin-3-O-glucoside, which can effectively control the content of 4-HNE, and can inhibit or eliminate 4-HNE. In particular, when cyanidin-3-O-glucoside and synephrine are used in combination, synephrine acts as a catalyst to promote cyanidin-3-O-glucoside to inhibit 4-HNE, and can effectively reduce the content of 4-HNE, thereby avoiding its further reaction with nucleophilic biological macromolecules to form various irreversible harmful addition or cross-linking products.

[0035] In the present invention, cyanidin-3-O-glucoside and synephrine are used as scavengers of 4-HNE, and can also directly play a role in natural forms such as mulberry, bayberry, tangerine peel, and orange to remove 4-HNE produced in the environment, food processing, and in the body, and further block the formation of harmful cross-linked products induced by 4-HNE, thereby preventing the harm caused to the environment and human body. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 The inhibition rate of cyanidin-3-O-glucoside on 4-HNE under different conditions (A is under simulated physiological conditions, B is under simulated food processing conditions); different lowercase letters indicate significant differences at different reaction times at the same concentration (p<0.05); different uppercase letters indicate significant differences at different concentrations at the same reaction time (p<0.05);

[0037] Figure 2 The reaction mechanism diagram of cyanidin-3-O-glucoside of the present invention with 4-HNE under simulated physiological conditions;

[0038] Figure 3 The mass spectrometry results of cyanidin-3-O-glucoside and its adduct with 4-HNE of the present invention; (Figure A is the ESI-MS of C3G 1 and MS 2 Mass spectrum; Figure B is the ESI-MS of C3G-HNE-(1-6) 1 and MS2 Mass spectra;

[0039] Figure 4 The effect of adding different proportions of synephrine on the capture of 4-HNE product by cyanidin-3-O-glucoside under the simulated processing conditions of the present invention; different lowercase letters indicate significant differences under different molar ratios (p<0.05);

[0040] Figure 5 The inhibition rate of synephrine and cyanidin-3-O-glucoside on 4-HNE under simulated processing conditions; different capital letters indicate significant differences between different substances at the same concentration (p<0.05); different lowercase letters indicate significant differences between the same substance at different concentrations (p<0.05);

[0041] Figure 6 The catalytic mechanism of synephrine and cyanidin-3-O-glucoside on 4-HNE when incubated for different times under simulated processing conditions. DETAILED DESCRIPTION

[0042] The experimental methods described in the examples are conventional methods unless otherwise specified; the reagents and materials described are commercially available unless otherwise specified.

[0043] Example 1

[0044] Purification and structural study of the adduct of cyanidin-3-O-glucoside and 4-HNE

[0045] (2) Experimental materials and instruments

[0046] Cyanidin-3-O-glucoside (C3G, purity >96%, Chengdu Ruifensi Biotechnology Co., Ltd.); 4-hydroxy-2-nonenal (4-HNE, 98%, chromatographic grade, Zhenjiang Diai Biotechnology Co., Ltd.); purified water (Hangzhou Wahaha Group Co., Ltd.); methanol, sodium dihydrogen phosphate, and sodium hydrogen phosphate were all analytical grade reagents (Shanghai Sinopharm Chemical Reagent Co., Ltd.).

[0047] Waters Xevo TQ-XS-LC-MS / MS (Waters, USA)

[0048] (2) Experimental procedures

[0049] ① Preparation of the product of cyanidin-3-O-glucoside and 4-HNE

[0050] 0.487 g of cyanidin-3-O-glucoside (C3G) was weighed and dissolved in 20 mL of phosphate buffer solution (PBS, 0.1 mol / L, pH=7.0), and a 4-NNE solution diluted with ethanol was added to react C3G and 4-HNE at a molar ratio of 1:3 at 100°C in the dark for 0.5 h. After terminating the reaction in an ice bath with 20 μL of acetic acid, the reaction solution was concentrated by rotary evaporation, and then the product was purified by a reverse phase ODS column (2.6 cm×30 cm) with a gradient elution of 10% to 35% methanol water. The 35% methanol water eluate was collected and freeze-dried to obtain the product C3G-HNE-1 (25 mg) of cyanidin-3-O-glucoside and 4-HNE.

[0051] ②Structural identification of C3G-HNE-1

[0052] C3G-HNE-1 was dissolved in methanol to obtain a 1 mg / mL standard solution, which was then diluted with methanol to a 500 ng / mL solution. The molecular weight was analyzed by UPLC-QTOF-MS / MS under the following conditions: 1D-NMR ( 1 H, 13 C), and 2D-NMR (HMQC, HMBC) was used for structural analysis.

[0053] a) Chromatographic conditions

[0054] Instrument: Waters Synapt-G2-Si

[0055] Chromatographic column: ACQUITY UPLC BEH C18 (1.7 μm, 2.1 × 100 mm, waters)

[0056] Injection volume: 1 μL; column temperature: 40°C; flow rate: 0.2 mL / min;

[0057] Mobile phase: 0.1% formic acid in water (phase A): methanol (phase B);

[0058] Program elution conditions: 0-1 min, 20% B; 1-2 min, 20-40% B; 2-5 min, 40-60% B; 5-8 min, 60-90% B; 8-10 min, 90% B; 10-10.1 min, 90-20% B; 10.1-13 min, 20% B.

[0059] b) Mass spectrometry conditions

[0060] Ionization mode: ESI+Data acquisition mode: MSe

[0061] Capillary voltage: 2.0kV Cone voltage: 20V Collision energy: 15-30eV

[0062] Ion source temperature: 120℃ Nebulizer gas temperature: 500℃

[0063] Cone gas flow rate: 50L / h Atomization gas flow rate: 1000L / h

[0064] Scan time: 3-10min

[0065] (3) Experimental results

[0066] ① C3G-HNE-1 structure identification, such as Figure 3 As shown in B, in positive ion mode, the parent ion mass is 605.2234 [M+H] + , which is 156 mass units (Mw 4-HNE ,156), fragment ion m / z 443[M+H] + ( Figure 3 B), presumably formed by loss of a glycoside; fragment ion m / z 425 [M+H] + , presumably due to the loss of a glycoside and then a water molecule; fragment ion m / z 287 [M+H] + , which is a typical fragment ion of C3G, is formed due to the loss of a glycoside molecule. It can be preliminarily indicated as the addition product of 4-HNE. 1 H and 13 C NMR data are shown in Table 1. As shown in Table 1, C3G-HNE-1 has similar carbon skeletons from C-1 to C-10 as cyanidin-3-O-glucoside. First, nine new signals were found (δC 61.58 and δH 5.56; δC 128.62 and δH 6.17; δC 133.11 and δH 5.60; δC 73.42 and δH 4.18; δC 37.59 and δH 1.46; δC 21.58 and δH 1.30; δC 32.19 and δH 1.25; δC 27.75 and δH 1.28; δC14.13 and δH 0.88), which may come from 4-HNE. In addition, one carbonyl signal of 4-HNE disappeared. At the same time, combined with the related signals from H11 to C-5, C-6, and C-7 in the HMBC spectrum, based on these characteristics, the structure of C3G-HNE-1 was finally determined to be a new adduct, and its structure is shown below.

[0067]

[0068] Table 1 C3G and its adduct product with 4-HNE C3G-HNE-1 1 H NMR (400 Hz) and 13 C NMR

[0069]

[0070]

[0071] Example 2

[0072] Inhibitory activity of cyanidin-3-O-glucoside on 4-HNE

[0073] (1) Experimental materials and instruments

[0074] Cyanidin-3-O-glucoside (purity>96%, Chengdu Ruifensi Biotechnology Co., Ltd.); 2,4-dinitrophenylhydrazine (DNPH, 98%, Shanghai TiCI Chemical Industry Development Co., Ltd.); 4-hydroxy-2-nonenal (4-HNE, 98%, chromatographic grade, Zhenjiang Diai Biotechnology Co., Ltd.); acetonitrile (chromatographic grade, Shanghai Sinopharm Chemical Reagent Co., Ltd.); purified water (Hangzhou Wahaha Group Co., Ltd.); sodium dihydrogen phosphate and sodium hydrogen phosphate were analytical grade reagents (Shanghai Sinopharm Chemical Reagent Co., Ltd.).

[0075] High performance liquid chromatograph: Waters 2695 (Waters Corporation, USA); ZQTY-70 desktop shaking incubator (Shanghai Zhichu Instrument Co., Ltd.); QL-861 vortex mixer (Qilin Bell Instrument Manufacturing Co., Ltd., Haimen City, Jiangsu Province); KQ-300B ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.); PHS-3C digital pH meter (Shanghai Sanxin Instrument Factory); FA2104N electronic analytical balance (Shanghai Precision Scientific Instrument Co., Ltd.).

[0076] (2) Experimental procedures

[0077] ① Inhibitory activity of cyanidin-3-O-glucoside against 4-HNE under simulated physiological conditions

[0078] Prepare 4-HNE solution with ethanol and C3G solution with methanol. In a 2mL centrifuge tube, add 0.1mL 5.0mmol / L 4-HNE and 0.1mL 20, 30, 50mmol / L C3G, then add 0.8mL 0.1mol / L, pH=7.4 PBS, vortex mix, and place in a 37℃, 220rpm shaker to react in the dark for 0, 0.5, 2, 4, 6, 8, 12, 24, 48, 72h, and immediately add 20μL acetic acid in an ice bath to terminate the reaction after sampling. In the blank control group, the same volume of methanol was used to replace the cyanidin-3-O-glucoside solution. At the same time, 500μL of the reaction solution was taken, and 300μL 1.25mg / mL DNPH solution was added to the shaker at 37℃, 220rpm for derivatization for 2h. The 4-HNE clearance rate was calculated by HPLC analysis. Three parallel groups were made for each sample.

[0079] ②Inhibitory activity of cyanidin-3-O-glucoside on 4-HNE under simulated processing conditions

[0080] Prepare 4-HNE solution with ethanol and C3G solution with methanol. In a 2mL centrifuge tube, add 0.1mL 5.0mmol / L 4-HNE and 0.1mL 10, 30, 50mmol / L C3G, then add 0.8mL 0.1mol / L, pH=7.0 PBS, vortex mix, place in a 100℃ water bath to avoid light for 5, 15, 30min, and immediately add 20μL acetic acid in an ice bath to terminate the reaction after sampling. For the blank control group, replace the cyanidin-3-O-glucoside solution with the same volume of methanol. At the same time, take 500μL of the reaction solution, add 300μL 1.25mg / mL DNPH solution, and derivatize for 2h at 37℃ and 220rpm in a shaker. 4-HNE clearance rate is calculated by HPLC analysis. Each sample is made in 3 groups in parallel.

[0081] ③HPLC method:

[0082] Waters 2695 high performance liquid chromatograph (Waters, USA) was used for detection, with Symmetry C18 column (250×4.6 mm, id5 μm), injection volume 10 μL, column temperature 30°C. Detection wavelength 372 nm, mobile phase A was acetonitrile, mobile phase B was ultrapure water (containing 0.1% formic acid), flow rate 1.0 mL / min, isocratic elution with 70% mobile phase A for 10 min.

[0083] ④Calculation formula for 4-HNE clearance rate

[0084]

[0085] (3) Data analysis

[0086] SPSS 27.0 statistical software (IBM, Armonk, NY, USA) was used for one-way analysis of variance (ANOVA), and Dunnett's multiple comparison test was used for statistical analysis. p < 0.05 indicated a significant difference. Different lowercase letters indicate significant differences at different reaction times at the same concentration (p < 0.05); different uppercase letters indicate significant differences at different concentrations at the same reaction time (p < 0.05).

[0087] (4) Experimental results

[0088] Depend on Figure 1A and 1B show that the scavenging activity of C3G on 4-HNE is time- and dose-dependent, whether under simulated physiological or simulated processing conditions. At 37°C, after 12 hours of reaction, the capture rate of C3G on 4-HNE can reach 50%, and after 72 hours of reaction, the inhibition rate of C3G on 4-HNE is as high as 79%. Under simulated processing conditions, the inhibitory activity of C3G is improved. After incubation at 100°C for 15 minutes, the addition of 5mmol / L C3G can clear about 50% of 4-HNE.

[0089] Example 3

[0090] Study on the scavenging mechanism of cyanidin-3-O-glucoside on 4-hydroxy-2-nonenal under simulated physiological conditions

[0091] (1) Experimental materials and instruments

[0092] Cyanidin-3-O-glucoside (purity>96%, Chengdu Ruifensi Biotechnology Co., Ltd.), 4-hydroxy-2-nonenal (4-HNE, 98%, chromatographic grade, Zhenjiang Diai Biotechnology Co., Ltd.); methanol, acetonitrile (chromatographic grade, Shanghai Sinopharm Chemical Reagent Co., Ltd.); purified water (Hangzhou Wahaha Group Co., Ltd.); sodium dihydrogen phosphate and disodium hydrogen phosphate were analytical grade reagents (Shanghai Sinopharm Chemical Reagent Co., Ltd.).

[0093] Waters Xevo TQ-XS triple quadrupole mass spectrometer (Waters, USA).

[0094] (2) Experimental procedures

[0095] Add 0.1 mL of C3G solution (50 mmol / L) prepared in methanol and 0.1 mL of 4-HNE solution (50 or 150 mmol / L) prepared in ethanol to the centrifuge tube, and then add 0.8 mL of PBS (0.1 mol / L, pH = 7.4) to make the molar concentration ratio of C3G:4-HNE = 1:1 and 1:3. After vortex mixing, place in a 37°C constant temperature shaker at 220 rpm, and shake in the dark for 5, 30, 120 and 480 min. Add 20 μL of acetic acid at each time point to terminate the reaction and cool in an ice bath. Take 100 μL of sample and dilute it with pure water, filter it through a 0.22 μm water filter membrane, and analyze it using UPLC-MS / MS.

[0096] ①UPLC conditions:

[0097] A Waters BEH C18 chromatographic column (100×2.1mm id, 1.7μm) was selected, the column temperature was 40°C, pure water containing 0.1% formic acid (eluent A); methanol (eluent B), flow rate was 0.3mL / min, elution conditions: 0-1min, 20% B; 1-2min, 20-40% B; 2-5min, 40-60% B; 5-8min, 60-90% B; 8-10min, 90% B; 10-10.1min, 90-20% B; 10.1-13min, 20% B; injection volume: 1μL.

[0098] ②Mass spectrometry conditions:

[0099] Ionization mode: ESI+Data acquisition mode: MRM

[0100] Capillary voltage: 2.0 kV Cone voltage: 20 V Collision energy: 15 eV

[0101] Ion source temperature: 120℃ Nebulizer gas temperature: 500℃

[0102] Cone gas flow rate: 50L / h Atomization gas flow rate: 1000L / h

[0103] Scan time: 3-10min

[0104] (3) Experimental results

[0105] Depend on Figure 2 It can be seen that at 37°C and pH 7.4, when C3G and 4-HNE react at a molar concentration ratio of 1:1 for only 5 minutes, three C3G-HNE products will appear. C3G-HNE-(1-3) (RT = 4.39, 4.90 and 5.56 min). The parent ion mass of the three is m / z 605 [M+H] + , 156 mass units higher than C3G (Mw 4-HNE, 156), fragment ion m / z 443 [M+H] + ( Figure 3 B), presumably formed by loss of a glycoside; fragment ion m / z 425 [M+H] + , presumably due to the loss of a glycoside and then a water molecule; fragment ion m / z 287 [M+H] + , which is a typical fragment ion of C3G, is formed due to the loss of a glycoside molecule ( Figure 3A). The results showed that the three compounds were isomers of the monoadduct of C3G and 4-HNE, named as C3G-HNE-1, C3G-HNE-2 and C3G-HNE-3. As the reaction time was extended to 30 min, two new product peaks appeared (RT = 6.02 and 6.30 min), and the parent ion mass was m / z 605 [M+H] + , 156 mass units higher than C3G (Mw4-HNE, 156), fragment ion m / z 443 [M+H] + ( Figure 3 B), presumably formed by loss of a glycoside; fragment ion m / z 425 [M+H] + , presumably due to the loss of a glycoside and then a water molecule, m / z 137[M+H] + It is a typical fragment ion of C3G ( Figure 3 A), which is formed by the breakage of the B and C rings after the loss of a glycoside molecule. The results show that these two compounds are isomers of the monoadduct of C3G and 4-HNE, named: C3G-HNE-4 and C3G-HNE-5. When the reaction time is extended to 120min, a new product peak appears (RT = 3.98min), and the fragment ion m / z 443 [M+H] + ( Figure 3 B), presumably formed by loss of a glycoside; fragment ion m / z 425 [M+H] + , presumably due to the loss of a glycoside and then a water molecule; fragment ion m / z 287 [M+H] + , which is a typical fragment ion of C3G, is formed due to the loss of a glycoside molecule. The results show that it is a single adduct of C3G and 4-HNE, named C3G-HNE-6. Therefore, C3G and 4-HNE can form 6 isomers of single adducts, and the amount of products increases with the increase of reaction time and reaction molar ratio. It can be seen that C3G can remove 4-HNE by capturing 4-HNE to form multiple adducts.

[0106] Example 4

[0107] Mechanistic study on the inhibition of 4-hydroxy-2-nonenal by cyanidin-3-O-glucoside catalyzed by synephrine under simulated processing conditions

[0108] (1) Experimental materials and instruments

[0109] Synephrine (98%, HPLC) (Shanghai Yuanye Biotechnology Co., Ltd.); cyanidin-3-O-glucoside (purity>96%, Chengdu Ruifensi Biotechnology Co., Ltd.); 2,4-dinitrophenylhydrazine (DNPH, 98%, Shanghai TiCI Chemical Industry Development Co., Ltd.); 4-hydroxy-2-nonenal (4-HNE, 98%, chromatographic grade, Zhenjiang Diai Biotechnology Co., Ltd.); methanol, ethanol, acetonitrile (chromatographic grade, Shanghai Sinopharm Chemical Reagent Co., Ltd.); purified water (Hangzhou Wahaha Group Co., Ltd.); sodium dihydrogen phosphate and sodium hydrogen phosphate were all analytical grade reagents (Shanghai Sinopharm Chemical Reagent Co., Ltd.).

[0110] Xevo TM TQ-XS triple quadrupole mass spectrometer (Waters, Inc., USA); ZQTY-70 desktop shaking incubator (Shanghai Zhichu Instrument Co., Ltd.); high performance liquid chromatograph: Waters 2695 (Waters, Inc., USA).

[0111] (2) Experimental steps and results

[0112] ① The promoting effect of different synephrine addition ratios on the scavenging of 4-HNE by cyanidin-3-O-glucoside

[0113] 1.1 Experimental procedures

[0114] Synephrine (SYN) and cyanidin-3-O-glucoside (C3G) were dissolved in methanol to obtain 50 mmol / L stock solutions, which were then diluted to corresponding concentrations with PBS (0.1 mol / L, pH = 7.0); the 4-HNE standard solution was diluted with ethanol to obtain a 4-HNE solution with a concentration of 1.0 mmol / L.

[0115] Compounded according to the molar ratio of SYN:C3G = 1:4 / 1:2 / 1:1 / 2:1

[0116] C3G alone: ​​add 1 mL of 4-HNE solution (1.0 mmol / L) and 1 mL of C3G solution into a glass explosion-proof tube, respectively, so that the final concentration of C3G is 2.0 mmol / L;

[0117] Combination of SYN and C3G: 1 mL of 4-HNE solution (1.0 mmol / L) and 1 mL of a mixed solution of C3G and SYN were added to glass explosion-proof tubes, respectively, so that the final concentration of C3G was 2.0 mmol / L, and the final concentrations of SYN were 0.5, 1.0, 2.0, and 4.0 mmol / L, respectively.

[0118] All the mixed solutions reacted with 4-HNE were vortexed and mixed, and then reacted in a 100°C constant temperature oil bath pot in the dark for 30 minutes. After the reaction was completed, 20 μL of acetic acid was added and immediately placed in an ice bath to terminate the reaction. After the reaction solution was diluted with methanol, it was filtered with a 0.22 μm organic membrane, and 1 μL was taken for LC-MS / MS analysis to detect the content of C3G and 4-HNE adducts. The detection method was the same as in Example 3, and all analyses were repeated three times.

[0119] 1.2 Experimental Results

[0120] from Figure 4 It can be seen that compared with C3G alone, the inhibitory effect of C3G on 4-HNE was significantly improved after adding SYN. ​​The amount of 4-HNE captured by C3G was 7-11 times that of C3G alone, and the amount of C3G-HNE-(1-6) product generated increased with the increase of SYN addition ratio, which shows that SYN plays the role of promoter or catalyst in binary combination. When the SYN:C3G molar ratio is 1:2, the amount of product is about 9.5 times that of C3G alone. After that, the amount of C3G-HNE-(1-6) generated increases slowly when SYN is continued to be added, so the SYN:C3G molar ratio = 1:2 was selected as the experimental condition.

[0121] ② Determination of the inhibitory activity of synephrine and cyanidin-3-O-glucoside at different dosages on 4-HNE

[0122] 2.1 Experimental procedures

[0123] Compound according to the molar ratio of SYN:C3G=1:2

[0124] SYN or C3G alone: ​​add 1 mL of 4-HNE solution (1.0 mmol / L) and 1 mL of SYN or C3G solution into a glass explosion-proof tube, respectively. The final concentrations of SYN are 0.25, 0.5, 1.0, 2.0 and 4.0 mmol / L, and the final concentrations of C3G are 0.5, 1.0, 2.0, 4.0, 8.0 mmol / L.

[0125] Combination of SYN and C3G: Add 1 mL of 4-HNE solution (1.0 mmol / L) and 1 mL of a mixed solution of SYN and C3G into a glass explosion-proof tube, respectively, so that the final concentrations of SYN and C3G are 0.25 and 0.5, 0.5 and 1.0, 1.0 and 2.0, 2.0 and 4.0, and 4.0 and 8.0 mmol / L, respectively.

[0126] All the mixed solutions reacted with 4-HNE were vortexed and mixed, and then reacted in a 100°C constant temperature oil bath pot in the dark for 30 minutes. After the reaction was completed, 20 μL of acetic acid was added and immediately placed in an ice bath to terminate the reaction. The blank control group used the same volume of methanol solution instead of SYN and C3G solution. 0.5 mL of each sample was derivatized according to the method in Example 2, filtered with a 0.22 μm organic filter membrane, and determined by HPLC. The detection method is the same as in Example 2. Three parallel samples were prepared for each sample.

[0127] 2.2 Experimental Results

[0128] At a fixed ratio, the inhibitory activity of SYN-catalyzed C3G on 4-HNE was studied at different concentrations. Figure 5 It can be seen that SYN has almost no inhibitory effect on 4-HNE ( Figure 5 The degradation effect of SYN alone was almost invisible in the results of the experiments. However, the inhibition rate of C3G on 4-HNE increased significantly with the increase of the amount of additives, whether used alone or in combination with SYN. ​​Moreover, compared with the single use, the inhibition rate of the compound group with SYN added on 4-HNE was 1.6-2.5 times that of the C3G group alone at 5 different doses, and the clearance rate of 4-HNE was significantly improved. After 30 minutes of reaction at 100°C, adding 1mmol / L C3G and 0.5mmol / L SYN could clear more than 50% of 4-HNE, which fully demonstrated that SYN was the catalyst in the process of C3G capturing 4-HNE.

[0129] ③Inhibitory activity of SYN and C3G on 4-HNE at different incubation times at fixed doses

[0130] 3.1 Experimental procedures

[0131] 0.5mL C3G solution (final concentration of 2mmol / L) and 0.5mL SYN solution (final concentration of 1mmol / L) were used alone or in combination with 4-HNE (final concentration of 0.5mmol / L) in equal volumes at 100°C in the dark for 5, 15, and 30min, and the inhibitor was replaced with the same volume of methanol as a blank. After the reaction was completed, 20μL of acetic acid was added and immediately placed in an ice bath to terminate the reaction. After the reaction solution was diluted with methanol and filtered with a 0.22μm organic filter membrane, 1μL was taken for LC-MS / MS analysis to detect the content of C3G and 4-HNE adducts. The detection method was the same as in Example 3, and all analyses were repeated three times.

[0132] 3.2 Experimental Results

[0133] The changes of adduct yield over time when SYN and C3G were used in combination at fixed doses were studied. Figure 6As shown, no SYN-captured 4-HNE product was found, while the amount of C3G-HNE-(1-6) product generated gradually increased with the extension of incubation time, and the amount of C3G-HNE-(1-6) product generated in the SYN-added group was much higher than the adduct yield when C3G was used alone. At each time point, the content of C3G-HNE-(1-6) in the C3G and SYN combined group was about 7-9 times higher than that when C3G was used alone (calculated by peak area), which proved from another perspective that SYN is a promoter or catalyst of C3G.

[0134] In summary, the present invention has clarified its synergistic pathway by studying the effects of the ratio of inhibitors C3G and SYN, the dosage added, and different reaction times on the clearance rate and adduct yield, that is, SYN, as a C3G promoter or catalyst, can significantly improve the 4-HNE capture activity and capture speed of C3G, and promote C3G to capture more 4-HNE to form more adduct products. Although C3G itself is not superior in the capture activity of 4-HNE, its capture performance can be greatly improved by adding a small amount of SYN in the present invention.

Claims

1. Cyanidin-3-O-glucose or cyanidin-3-O-glucose combined with synephrine is used in the scavenging or inhibiting of 4-hydroxy-2-nonenal.

2. The use according to claim 1, characterized in that: The synephrine catalyzes cyanidin-3-O-glucose in inhibiting or removing 4-hydroxy-2-nonenal.

3. The use according to claim 1, characterized in that: The application of cyanidin-3-O-glucose or cyanidin-3-O-glucose combined with synephrine in inhibiting 4-hydroxy-2-nonenal in an environment.

4. The use according to claim 1, characterized in that: The application of cyanidin-3-O-glucose or cyanidin-3-O-glucose combined with synephrine in inhibiting 4-hydroxy-2-nonenal produced in food processing.

5. The use according to claim 1, characterized in that: The application of cyanidin-3-O-glucose or cyanidin-3-O-glucose combined with synephrine in removing 4-hydroxy-2-nonenal in a living body.

6. The use according to claim 1, characterized in that: The cyanidin-3-O-glucose or the combination of cyanidin-3-O-glucose and synephrine is used in the preparation of 4-hydroxy-2-nonenal inhibitors in the environment, food processing or in organisms to inhibit various harmful addition or cross-linking products formed by the reaction of 4-hydroxy-2-nonenal with nucleophilic biological macromolecules.

7. The use according to claim 1, characterized in that: The cyanidin-3-O-glucose or the cyanidin-3-O-glucose combined with synephrine is used to capture 4-hydroxy-2-nonenal to form an addition product, thereby reducing the content of 4-hydroxy-2-nonenal.

8. The use according to claim 1, characterized in that: The usage amount of cyanidin-3-O-glucose is preferably 0.5-8.0 mmol / L; the usage amount of synephrine is 0.25-4.0 mmol / L, and the usage amount of synephrine is 0-2 times the molar amount of cyanidin-3-O-glucose.

9. A 4-hydroxy-2-nonenal inhibitor in the environment, food processing or in an organism, characterized in that: The inhibitor is a preparation formed by using cyanidin-3-O-glucoside or cyanidin-3-O-glucoside and synephrine as the sole or main components.

10. The use according to claim 1 or the inhibitor according to claim 9, characterized in that: The cyanidin-3-O-glucoside is in the natural form of mulberry or bayberry; synephrine can directly act in the natural form of tangerine peel, orange or tangerine flower.

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