阅读说明：本技术 矢车菊素-3-o-葡萄糖苷及其与丙烯醛加合产物作为丙烯醛抑制剂的应用 (cyanidin-3-O-glucoside and application of addition product of cyanidin-3-O-glucoside and acrolein as acrolein inhibitor ) 是由 吕丽爽 宋小莉 卢永翎 司波 于 2021-07-23 设计创作，主要内容包括：本发明公开了矢车菊素-3-O-葡萄糖苷或其与丙烯醛加合产物作为丙烯醛抑制剂的应用,本发明公开了矢车菊素-3-O-葡萄糖苷及其与丙烯醛的一加合产物或者二加合产物的一种新应用,即其能够快速有效捕获ACR以控制其含量,避免其与生物大分子内的亲核基团如氨基、巯基、羟基和咪唑基结合,形成交联产物,妨碍机体功能正常行使。本发明的矢车菊素-3-O-葡萄糖苷或其与丙烯醛的加合产物如C3G-ACR可继续作为ACR的清除剂或者抑制剂,清除或者抑制环境中、食品加工中的丙烯醛,同时可以清除进入体内的外源性ACR或人体内源性产生的ACR,从而控制或缓解由ACR引起的疾病症状。(The invention discloses application of cyanidin-3-O-glucoside or an addition product of the cyanidin-3-O-glucoside and acrolein as an acrolein inhibitor, and discloses new application of the cyanidin-3-O-glucoside and a mono-addition product or a di-addition product of the cyanidin-3-O-glucoside and the acrolein, namely the new application can quickly and effectively capture ACR to control the content of the ACR and prevent the ACR from being combined with nucleophilic groups such as amino, sulfhydryl, hydroxyl and imidazolyl in biological macromolecules to form a cross-linked product to prevent normal running of body functions. The cyanidin-3-O-glucoside or the addition product of the cyanidin-3-O-glucoside and the acrolein, such as C3G-ACR, can be continuously used as a scavenger or an inhibitor of ACR, can scavenge or inhibit the acrolein in the environment and food processing, and can scavenge exogenous ACR entering the body or ACR endogenously generated by the human body, thereby controlling or relieving the disease symptoms caused by the ACR.)

1. The use of cyanidin-3-O-glucoside or an addition product thereof with acrolein as an acrolein inhibitor, the addition product comprising a mono-addition product C3G-ACR or a di-addition product C3G-2ACR of cyanidin-3-O-glucoside and acrolein, the structures of which are respectively shown below:

2. use according to claim 1, characterized in that the cyanidin-3-O-glucoside or the addition product thereof with acrolein is used for the preparation of an inhibitor for inhibiting acrolein in an environment.

3. Use of cyanidin-3-O-glucoside or the addition product thereof with acrolein in the manufacture of an inhibitor for inhibiting acrolein produced in food processing according to claim 1.

4. Use according to claim 1, characterized in that the cyanidin-3-O-glucoside or the addition product thereof with acrolein is used in the preparation of an inhibitor for inhibiting acrolein in an organism.

5. Use according to claim 1, characterized in that the cyanidin-3-O-glucoside or its addition product with acrolein inhibits the use of various harmful addition or cross-linking products formed by the reaction of acrolein with nucleophilic biomacromolecules in manufacturing environments, food processing or in vivo acrolein inhibitors.

6. Application of cyanidin-3-O-glucoside or an addition product of cyanidin-3-O-glucoside and acrolein in preparing a medicament for preventing human chronic diseases.

7. Use according to claim 6, wherein the human chronic disease preferably comprises Alzheimer's disease, Parkinson's disease, rheumatoid arthritis or cardiovascular and cerebrovascular diseases.

8. The structures of the mono-addition product C3G-ACR and the di-addition product C3G-2ACR of cyanidin-3-O-glucoside and acrolein are respectively shown as follows:

9. an acrolein inhibitor in environment, food processing or organism, characterized in that the inhibitor is a preparation formed by using cyanidin-3-O-glucoside or an addition product thereof and acrolein as the only component or being used as a main component and being compounded and used together with other substances.

10. The application of myricetin in preparing an inhibitor for inhibiting acrolein generated in food processing is disclosed, wherein the main component of myricetin is cyanidin-3-O-glucoside.

Technical Field

The invention belongs to the field of anthocyanin application, and particularly relates to cyanidin-3-O-glucoside and application of an addition product of the cyanidin-3-O-glucoside and acrolein as an acrolein inhibitor.

Background

ACR is a highly toxic unsaturated aldehyde that is widely found in external environments, such as cigarettes, fossil fuels (gasoline or petroleum), building fires; secondly, it can also be produced by food processing and human endogenous metabolism. ACR emissions from automobile exhaust are reported to be 1.8 tons/year, whereas ACR emissions in commercial kitchen fumes can be as high as 7.7 tons/year. In the brewing process of alcoholic beverages, ACR can be generated by means of microbial metabolism, for example, the average concentration of ACR in finished white spirit can reach 72.3 mug/L, the ACR concentration range in whisky is 700-.

In the food processing process, ACR is mainly generated by the thermal degradation of carbohydrate and amino acid, the cracking of fatty acid, the fermentation and metabolism of microorganism and the like; wherein the research data indicate that the ACR content in the fried and baked food is generally higher, ranging from 0.001 to 0.9 mg/kg. In humans, ACR can be produced endogenously by enzyme-mediated oxidation of polyamines or threonine, by oxidation of unsaturated fatty acids with Reactive Oxygen Species (ROS), and by microbial metabolism in the human gut.

ACR, a highly water-soluble α, β -unsaturated aldehyde, rapidly enters physiological tissues. And has high reactivity with cell nucleophiles (such as protein, DNA and RNA) due to strong electrophilicity. Reacting with cysteine, histidine and lysine residues in protein to influence the antigen recognition capability of immunoglobulin, thereby causing autoimmune diseases (sjogren's syndrome and rheumatoid arthritis); reacts with guanine base in DNA to generate a cyclic addition product, which causes DNA damage to further cause carcinogenesis and mutation; ACR can also cause oxidative stress, leading to neuronal damage.

Therefore, the method finds natural, safe and efficient substances for capturing the ACR so as to reduce the level of the ACR in the environment or in vivo, researches the mechanism of the ACR, is one of necessary means for preventing environmental pollution, improving food safety and preventing and treating chronic diseases, and has important practical significance and theoretical value.

cyanidin-3-O-glucoside (C3G) is a anthocyanin, belongs to a flavonoid compound, is widely present in colored grains, fruits and vegetables such as waxberries, blackberries, purple cabbages, black rice and the like, has good water solubility and good physiological activity such as oxidation resistance, anti-inflammation, bacteriostasis and the like, and has not been found to be researched on the aspect of inhibiting acrolein.

The prior art discloses a reaction mechanism of cyanidin-3-O-glucoside capture of 1, 2-dicarbonyl compounds (glyoxal, GO), which is the direct addition of glyoxal and C3G, and can only form a di-addition product; but acrolein is far more toxic than glyoxal; there is therefore a need to develop new acrolein inhibitors.

Disclosure of Invention

The purpose of the invention is as follows: aiming at the problems in the prior art, the invention provides cyanidin-3-O-glucoside and application of an addition product of the cyanidin-3-O-glucoside and acrolein as an acrolein inhibitor, wherein the cyanidin-3-O-glucoside and the addition product of the cyanidin-3-O-glucoside and the acrolein can inhibit the acrolein existing in a natural environment solution system and can also solve the problem of the acrolein generated in a normal-temperature food processing system; and inhibiting acrolein in organisms.

The invention also provides an addition product of cyanidin-3-O-glucoside and acrolein, and an acrolein inhibitor in the environment or in an organism.

The technical scheme is as follows: in order to achieve the above object, the cyanidin-3-O-glucoside and the addition product thereof and acrolein are used as an acrolein inhibitor; the addition product comprises a mono-addition product C3G-ACR or a di-addition product C3G-2ACR of cyanidin-3-O-glucoside and acrolein, and the structures of the addition products are respectively shown as follows:

wherein, the application of the cyanidin-3-O-glucoside and acrolein addition product in the preparation environment of an acrolein inhibitor.

Further, the cyanidin-3-O-glucoside and the addition product thereof with acrolein can capture acrolein so as to reduce the content of the acrolein.

Wherein the cyanidin-3-O-glucoside or the addition product thereof and acrolein is used for preparing an inhibitor for inhibiting the generation of acrolein in food processing. In a normal-temperature food processing system, cyanidin-3-O-glucoside is used as a anthocyanin contained in food raw materials such as fruits and vegetables, can capture acrolein in the food processing process to form C3G-ACR, and the addition product can continuously capture the acrolein, so that the content of the acrolein in the food is greatly reduced, the blank of an acrolein inhibitor in the normal-temperature food system such as fruit wine and jelly production is filled, for example, a large amount of C3G is contained in waxberries, and when related waxberry products are produced, if the acrolein is generated, the C3G can capture the acrolein.

Wherein, the cyanidin-3-O-glucoside or the addition product thereof and the acrolein are applied to the preparation of the inhibitor for inhibiting the acrolein in the organism.

Further, the use of the cyanidin-3-O-glucoside or its addition product with acrolein to inhibit the formation of various deleterious addition or cross-linking products formed by the reaction of acrolein with nucleophilic biomacromolecules in manufacturing environments, food processing, or in vivo acrolein inhibitors.

The invention relates to the application of cyanidin-3-O-glucoside or the addition product of cyanidin-3-O-glucoside and acrolein in preparing medicaments for preventing human chronic diseases.

Wherein, the human chronic diseases comprise Alzheimer's disease, Parkinson's disease, rheumatoid arthritis or cardiovascular and cerebrovascular diseases.

The cyanidin-3-O-glucoside or the addition product thereof and the acrolein can be prepared into related reagents or medicaments for inhibiting or eliminating the acrolein in the environment, food processing or organisms.

The cyanidin-3-O-glucoside and the mono-addition product and the di-addition product of the cyanidin-3-O-glucoside and the acrolein are as follows: respectively C3G, C3G-ACR and C3G-2ACR, and the structures are respectively shown as follows:

the inhibitor is a preparation formed by taking cyanidin-3-O-glucoside or an addition product of the cyanidin-3-O-glucoside and acrolein as the only component or taking the inhibitor as the main component and compounding and using the inhibitor and other substances together.

The invention relates to an application of red bayberry in preparing an inhibitor for inhibiting acrolein generated in food processing, wherein the red bayberry mainly comprises cyanidin-3-O-glucoside.

Wherein the cyanidin-3-O-glucoside is a main anthocyanin substance in fruits and vegetables.

The parent ring structure of cyanidin-3-O-glucoside is 2-phenyl benzopyran cation.

Aiming at acrolein with high toxicity in environment, food processing or organisms, an addition product formed after ACR is captured by C3G has the activity of continuously capturing ACR, and the reaction mechanism of capturing ACR by C3G is Michael addition reaction, namely hydroxyl on the ring A of the C3G structure reacts with aldehyde on the acrolein to form a hemiacetal structure after the C3G and ACR undergo 1, 4-addition reaction under alkaline conditions.

The ACR activity is continuously captured by the addition product formed after the ACR is captured by the C3G, and the acrolein capturing activity of the mono-addition product of the C3G and the ACR is higher than that of the C3G and the di-addition product of the C3G-2 ACR. Effectively fills the blank of acrolein inhibitor in the environment, normal temperature food processing system on the market and in organisms.

Has the advantages that: compared with the prior art, the invention has the following advantages:

the invention provides a new application of cyanidin-3-O-glucoside or an addition product of the cyanidin-3-O-glucoside and acrolein for the first time, which can effectively control the content of ACR and avoid various irreversible harmful addition or crosslinking products formed by further reaction of the ACR and nucleophilic biological macromolecules. cyanidin-3-O-glucoside and addition products of C3G-ACR with acrolein can be used as scavenger or inhibitor of ACR, and can scavenge acraldehyde in environment and food processing, and ACR produced exogenously or endogenously in vivo, and further block formation of harmful cross-linking products induced by ACR, and prevent harm to environment and human body.

Drawings

FIG. 1 is a graph comparing inhibition of ACR activity by cyanidin-3-O-glucoside and its addition products under simulated room temperature conditions;

FIG. 2 shows the chemical structures and mass spectra of cyanidin-3-O-glucoside and its addition products in accordance with the present invention; (FIG. A, FIG. B are ESI-MS of C3G, respectively¹And MS²A spectrogram; FIG. C and FIG. D are ESI-MS of C3G-ACR, respectively¹And MS²A spectrogram; FIG. E, FIG. F are ESI-MS for C3G-2ACR, respectively¹And MS²Spectrogram)

FIG. 3 shows the results of studies on the mechanism of elimination of acrolein by the cyanidin-3-O-glucoside-acrolein mono-adduct; (FIGS. A and B are liquid chromatogram of reaction of C3G-ACR with ACR for 1 and 5min, respectively)

FIG. 4 is a liquid phase-mass spectrum of C3G after C3G in red bayberry has reacted with acrolein in white spirit and its adduct product with acrolein;

FIG. 5 shows the measurement result of the activity of cyanidin-3-O-glucoside in inhibiting acrolein in fruit wine (the bar chart is C3G, C3G-ACR and C3G-2ACR from left to right).

Detailed Description

The experimental methods described in the examples are all conventional methods unless otherwise specified; the reagents and materials are commercially available, unless otherwise specified.

Example 1

Purification and structural study of cyanidin-3-O-glucoside and ACR addition product

(1) Experimental materials and instruments

Polyamide resin 100-; ethanol (analytical grade, Shanghai national drug group chemical Co., Ltd.); purified water (Hangzhou Wahaha group Co., Ltd.); hydrochloric acid (Shanghai national drug group chemical reagent Co., Ltd.)

AVANCE 400MHz nuclear magnetic resonance instrument (bruker corporation); 1290/6460 LC-MS (Agilent, USA).

(2) Experimental procedure

134.7mgC3G was weighed out and dissolved in 2mL of methanol, and 208 μ of LACR stock solution was removed and added to a 1.792mL pbs (PH 7.0, 0.1mol/L) solution so that the final molar ratio of C3G to ACR was 1: and 10, after vortex mixing, putting the mixture into a constant-temperature shaking table at 37 ℃ and 220rpm for reaction for 30min, taking out the sample, and cooling the sample in an ice bath for later use.

Keeping the molar concentration of C3G unchanged, ensuring that the molar ratio of the C3G to ACR is 1:3, uniformly mixing by vortex, placing the mixture into a constant-temperature shaking table at 37 ℃ and 220rpm for reaction for 60min, taking out a sample, and cooling the sample by ice bath for later use.

Loading the concentrated C3G and ACR reaction liquid according to the loading proportion of 1g of polyamide filler per 100g, after loading, carrying out gradient elution by water and 10% ethanol water (pH is 3), collecting eluent by a step collector, tracking and analyzing each tube of product by adopting polyamide thin-film chromatography until no product is detected, stopping elution, combining elution components with the same Rf value, carrying out rotary evaporation concentration, and storing in a refrigerator at-80 ℃.

Different molar ratios have been investigated, and it has been found that the amount of adduct produced is different, i.e.more mono-adduct at 1:3 and more di-adduct at 1: 10.

The first addition product C3G-ACR and the second addition product C3G-2ACR of C3G and ACR are obtained by respectively adopting the molar ratio of 1:3 and 1: 10. LC-MS analysis of molecular weight, 1D-NMR: (¹H，¹³C) 2D-NMR (HMQC, HMBC) for structural analysis.

(3) Results of the experiment

C3G-ACR identification

The mass of the parent ion of the prepared C3G-ACR is M/z 505[ M + H ] under the positive ion mode through the measurement of liquid chromatography-mass spectrometry]⁺Mass of parent ion M/z 449[ M + H ] to C3G]⁺56 (MW) in excess_ACR56) and the major fragment ion peak in MS/MS was M/z 343[ M + H ]]⁺Indicating the loss of one glucose molecule (m/z 162) and the fragment ion peak m/z 287 of C3G plus one molecule ACR (MW)_ACR56) The other major fragment ion peak is M/z299[ M + H ]]⁺Indicating the loss of one glucose molecule (m/z 162) and one [ -CHOH-CH2-](m/z 44) group, which indicates that C3G-ACR is an addition product of one molecule of ACR and C3G, of C3G-ACR¹H NMR (400Hz) and¹³the C NMR (100MHz) spectral data are specified in Table 1.

TABLE 1 addition product of C3G with C3G-ACR¹H NMR (400Hz) and¹³c NMR (100MHz) spectral data (deuterated methanol, in units of. delta. ppm)

As can be seen from Table 1, C3G-ACR has the same C-ring structure as C3G. The hydrogen signal at C3G H-6 disappeared, while 3 new hydrogen signals, δ, appeared at C3G-ACR_H 2.81(2H,d),δ_H2.17(2H, d) and δ_H5.82(1H, d), in addition HMBC mapping results show (FIG. 2), H-11 and δ_C 152.27(C-7),δ_C105.76(C-6),δ_C95.36(C-13) and delta C27.58 (C-12) are both related, and one-CHCH is judged to be in the C3G-ACR₂CH₂The side chain, attached to the A ring, in C3G-ACR the carbonyl group on the acrolein forms a hemiacetal structure by dehydrocondensation with the hydroxyl group on C-7. HPLC-MS/MS of the comprehensive C3G-ACR,¹H NMR、¹³The results of C NMR, HMBC and HMQC spectrograms (figure 2) finally determine the structure of C3G-ACR, and the compound is a novel compound and has the structure:

C3G-ACR identification

The peak of the prepared C3G-ACR excimer ion is 561[ M + H ] under positive ion mode ESI-MS (M/z) determined by LC-MS]⁺It was shown to have a molecular weight of 561, 112(2 MW) more than the molecular weight of C3G_ACR). The secondary mass spectrogram has fragment ion peak M/z 399[ M-162 [)]⁺，355[M-162-44]⁺，311[M-162-44-44]⁺One glucose molecule (m/z 162) and one [ CHOH-CH ] are lost by C3G-2ACR respectively₂-]Group (m/z 44), two [ CHOH-CH ]₂-]The radicals are formed. Thus, C3G-2ACR is presumed to be the di-adduct of C3G reacting with ACR. Of C3G-2ACR¹H NMR (400Hz) and¹³the C NMR (100MHz) spectral data are specified in Table 2.

TABLE 2 addition of C3G with C3G-2ACR¹H NMR (400Hz) and¹³c NMR (100MHz) spectral data (deuterated methanol, in units of. delta. ppm)

Comparing the hydrogen and carbon spectra of C3G-2ACR with C3G, C3G-2ACR has the same C ring structure as C3G. The hydrogen signals on the original C3G H-6 and H-5' disappear, and 6 new hydrogen signals delta appear_H 2.71(H-11，m)、δ_H 2.16(H-12，d)、δ_H 5.86(H-13，s)、δ_H 2.81(H-14，m)、δ_H2.03(H-15，d)、δ_H5.80(H-16, s), in addition HMBC mapping results show H-11 and δ_C 102.58(C-6)、δ_C93.63(C-13) has associations of H-14 with. delta_C 112.69(C-5′)，δ_C25.63(C-15) and δ_C93.59(C-16), it was concluded that there are two side chains-CHCH 2CH 2-in C3G-2ACRLinked to the A and B rings, respectively, to form two hemiacetal structures at the C3G C-6 and C-5' positions, respectively. Synthesis of C3G-2ACR¹H NMR、¹³And C NMR, HMBC and HMQC spectrogram results finally determine the structural formula of C3G-2ACR as follows:

example 2

The result of the measurement of ACR activity inhibition by cyanidin-3-O-glucoside and the addition product thereof with acrolein under the condition of simulating room temperature.

(1) Experimental materials and instruments

cyanidin-3-O-glucoside (purity > 85%, genderesin biotechnology limited); 2, 4-dinitrophenylhydrazine (DNPH & HCl, purity > 98%, Tokyo Chemical Industry); cyanidin-3-O-glucoside-acrolein mono-adduct (prepared in example 1); cyanidin-3-O-glucoside-acrolein bis-adduct product (prepared in example 1); acrolein (ACR, 98% aqueous solution, analytical purity, Shandong-Xiya chemical industries, Ltd.); acetonitrile (chromatographically pure, Shanghai national drug group chemical reagent, Inc.); purified water (Hangzhou Wahaha group Co., Ltd.); both sodium dihydrogen phosphate and disodium hydrogen phosphate were analytical reagents (Shanghai pharmaceutical group chemical Co., Ltd.).

High performance liquid chromatograph: agilent Technologies 1260 (Agilent Technologies, USA); ZQTY-70 desktop shake culture tank (Shanghai Zhichu instruments Co., Ltd.); QL-861 vortex mixer (Lenbel instruments, Inc. of Haiman city, Jiangsu); KQ-300B ultrasonic cleaner (Kunshan ultrasonic instruments Co., Ltd.); PHS-3C digital pH meter (Shanghai Sanxin Meter factory); FA2104N electronic analytical balance (shanghai precision scientific instruments ltd);

(2) experimental procedure

An ACR solution was prepared using 0.1mol/L PBS at pH 7.0, and a C3G, C3G-ACR, C3G-2ACR solution was prepared using methanol. Adding 0.5mL of 1.0mmol/LACR and 0.5mL of 3.0mmol/L C3G or C3G-ACR or C3G-2ACR into a 2mL centrifuge tube respectively, vortex uniformly, placing the mixture in a shaker at 25 ℃, reacting for 0, 15, 30, 60 and 120min respectively at 220rpm, and immediately stopping the reaction in ice bath after sampling. In the blank control group, PBS with the same volume is used for replacing cyanidin-3-O-glucoside solution, and 500 mu L of reaction liquid is simultaneously used for derivatization, and the ACR clearance rate is calculated through HPLC analysis. Each sample was run in 3 replicates.

HPLC method:

the separation detection is carried out by adopting Agilent 1260 high performance liquid chromatograph, and Kromasil 100-5C 18 chromatographic column (250 x 4.6mm i.d., 5 μm) and Diode Array Detector (DAD) are selected, the sample volume is 20 μ L, the column temperature is 30 ℃, the detection wavelength is 372nm, the mobile phase A is acetonitrile, the mobile phase B is ultrapure water (containing 0.1% formic acid), and the elution is carried out at 70% of the mobile phase A for 7.5min at the flow rate of 1.0mL/min in an isocratic manner.

(3) Results of the experiment

As can be seen from FIG. 1, under the simulated room temperature condition, the inhibitory activity of C3G-ACR on acrolein in the solution is much higher than the inhibitory effect of C3G on ACR, and the capture rate of C3G-ACR on ACR is up to 66% at the reaction time of 5min, while the capture rate of C3G and C3G-2ACR is almost 0. When the reaction time is 30min, the capture efficiency of C3G-ACR to ACR is still higher than that of C3G and C3G-2ACR to ACR, C3G can generate C3G-ACR after the reaction time is continued for 60min, and generated C3G-ACR can also continue to capture ACR and even generate a di-addition product C3G-2ACR, so the capture efficiency begins to increase.

Example 3

Research on elimination mechanism of acrolein by cyanidin-3-O-glucoside-acrolein-addition product

(1) Experimental materials and instruments

cyanidin-3-O-glucoside-acrolein mono-adduct product (prepared in example 1), acrolein (ACR, 98% aqueous solution, analytically pure, santo chekia chemical industries, ltd); acetonitrile (chromatographically pure, Shanghai national drug group chemical reagent, Inc.); purified water (Hangzhou Wahaha group Co., Ltd.); both sodium dihydrogen phosphate and disodium hydrogen phosphate were analytical reagents (Shanghai pharmaceutical group chemical Co., Ltd.).

High performance liquid chromatograph: agilent Technologies 1260 (Agilent Technologies, USA); ZQTY-70 desktop shake culture tank (Shanghai Zhichu instruments Co., Ltd.); QL-861 vortex mixer (Lenbel instruments, Inc. of Haiman city, Jiangsu);

(2) experimental procedure

Preparing ACR solution (15mM) with PBS 0.1M, pH 7.0.0, and preparing 5mM cyanidin-3-O-glucoside-acrolein mono-adduct (C3G-ACR) with methanol; 0.5mL of ACR solution and 0.5mL of C3G-ACR solution were incubated at 25 ℃ in a desktop shaking incubator at 220rpm for 1, 5min in the absence of light for liquid phase analysis.

(3) Results of the experiment

When C3G-ACR and ACR react for 1min in a molar ratio of 1:3, as shown in FIG. 3, C3G-2ACR appears in the system, which shows that C3G-ACR can eliminate ACR in the system by capturing ACR to generate an addition product, and the capture speed is very rapid. The content of C3G-2ACR generated after 5min of reaction can account for 40% of the whole reaction system. The capture activity of an addition product on ACR in a C3G-ACR simulated environment solution system is good, and one molecule of ACR is captured to form C3G-2ACR, so that the mechanism of capturing ACR by C3G is completely clarified.

Example 4

Determination of activity of myricetin and cyanidin-3-O-glucoside for inhibiting acrolein in fruit wine

(1) Experimental materials and instruments

Red bayberry (suzhou si yuan natural products limited); white spirit (Jiangsu peach forest wine industry Co., Ltd.); xevo^TMTQ-XS triple quadrupole mass spectrometer (Vorte Co., Ltd., USA)

(2) Experimental procedure

According to GB 31622-; the addition amount of the red bayberry in the fruit wine can reach 200mg/L according to GB 2760-2014 'standard for use of food additives in national standards for food safety'. Therefore, a fruit wine system is simulated, 2mg of myrica rubra is weighed and dissolved in 10mL of white wine (the alcoholic strength is 42 percent of common white wine), the white wine is placed for 1h, 4h, 24h, 3d and 5d, and the contents of C3G, C3G-ACR and C3G-2ACR in the system are respectively measured.

UPLC conditions:

a chromatographic column: ACQUITYBEH C18 column (1.7 μm, 2.1X 50mm, Waters); column temperature: 40 ℃; mobile phase: 0.1% aqueous formic acid (a), acetonitrile (B); gradient elution: 5% B, 0-2 min; 5-35% of B, 2-5 min; 5-35% of B, 5-5.1 min; 95% B for 5.1-6 min; 5% B, 6-8min. sample size: 10 μ L, flow rate: 0.4mL/min

MS/MS conditions

Using a Waters ACQUITY Xevo TQ-XS UPLC/MS system, electrospray ion source (ESI), positive ion mode, capillary voltage: 2.5kv, desolvation gas temperature: 500 ℃; the multiple reaction detection scan mode (MRM) was chosen, and the excimer ion peak, the major daughter ion fragment peak, and their acquisition parameters for the three species are shown in table 3.

TABLE 3 Mass Spectrometry acquisition parameters for C3G, C3G-ACR and C3G-2ACR

(3) Results of the experiment

The main component of red bayberry is C3G, as shown in figure 4, during the simulated brewing process of the red bayberry wine, the red bayberry wine can be kept for 1h to capture acrolein in the wine, C3G-ACR and C3G-2ACR are generated, and the content of the adduct is increased with the prolonging of the keeping time (as shown in figure 5). The red bayberry can be used as a main component of a coloring agent for food, and can also be used as an inhibitor or a scavenger of acrolein, and the C3G in the red bayberry can be used as a main component of the coloring agent for food, and can also be used as an inhibitor or a scavenger of acrolein, so that the red bayberry is beneficial to ecology or human health.