CN116439344B

CN116439344B - Application of synephrine or hesperidin and synephrine binary combination as acrolein inhibitor

Info

Publication number: CN116439344B
Application number: CN202310231997.0A
Authority: CN
Inventors: 司波; 吕丽爽; 梁雨; 仲宇晴
Original assignee: PRODUCT QUALITY SUPERVISING AND INSPECTING INSTITUTE OF SUQIAN CITY
Current assignee: PRODUCT QUALITY SUPERVISING AND INSPECTING INSTITUTE OF SUQIAN CITY
Priority date: 2023-03-10
Filing date: 2023-03-10
Publication date: 2024-03-29
Anticipated expiration: 2043-03-10
Also published as: CN116439344A

Abstract

The invention discloses application of synephrine or binary combination of hesperidin and synephrine as an acrolein inhibitor, and provides inhibition effect of the hesperidin or the synephrine on the acrolein for the first time, in particular application of the synephrine as an accelerator or a catalyst for synergistically inhibiting the acrolein with the hesperidin when the synephrine and the synephrine are used in a binary combination mode. The invention provides a new application of hesperidin and synephrine, namely binary combination of hesperidin and synephrine can rapidly and effectively capture acrolein in the environment or in food processing to control the content of the acrolein, avoid covalent bonding of the acrolein and nucleophilic sites of biological macromolecules in vivo such as DNA, protein, nucleotide, phospholipid and the like, further form various harmful crosslinking or addition products, and prevent the damage of the acrolein to human bodies.

Description

Application of synephrine or hesperidin and synephrine binary combination as acrolein inhibitor

Technical Field

The invention belongs to the application field of flavone and alkaloid, and relates to application of synephrine or binary combination of hesperidin and synephrine as an acrolein inhibitor.

Background

Acrolein (ACR) is a highly toxic unsaturated reactive carbonyl compound, listed as a class 3 carcinogen by the international agency for research on cancer (IARC) in 1995, and is a precursor of class 2A carcinogen acrylamide. Because the structure of the ACR contains carbonyl and vinyl, the ACR has higher reactivity and can be covalently combined with nucleophilic sites of DNA, protein, nucleotide and phospholipid, thereby causing a series of pathological reactions. In addition, ACR can exert toxic effects by inducing oxidative stress, disrupting organelles, and altering cellular signaling, among other indirect mechanisms. At present, the research shows that ACR can cause a series of chronic diseases such as cardiovascular diseases, atherosclerosis, alzheimer disease, tumors and the like. In addition to contaminated air, water sources, cigarette smoke, etc., one of the most important sources of exogenous ACR is dietary intake. Notably, ACR is widely found in environments such as aqueous solutions and various processed foods, including fried foods, baked goods, fermented foods, salted foods, alcoholic beverages, and the like. ACR is mainly derived from maillard reactions, lipid peroxidation, amino acid degradation, carbohydrate pyrolysis and microbial metabolism during food processing. Studies have shown that ACR levels in fat, protein and carbohydrate rich fractions are higher, such as 51+ -3 μg/kg and 93+ -5 μg/kg in grilled steak and grilled sausage, respectively, while ACR levels in bread and cheese are as high as 161+ -21 μg/kg and 1000 μg/kg, respectively. The daily maximum intake of ACR in daily life of adults is statistically about 0.1mg/kg body weight/day, whereas World Health Organization (WHO) prescribes a daily tolerance intake (TDI) of ACR of 7.5 μg/kg body weight/day, which is far in excess of TDI. Therefore, reducing ACR content in the environment is of great importance.

Although various strategies have been used to address this safety issue, the use of natural compounds as inhibitors is still considered to be most applicable. Certain phenolic compounds, amino acids and sulfur-containing compounds have proven to be effective ACR scavengers both in vitro and in vivo. However, some existing inhibitors have poor solubility, are unstable when exposed to heat, and have poor inhibition effects.

Hesperidin (HES), a flavonoid glycoside, is widely distributed in fruits and plants, such as lemon, orange, tangerine peel, seville orange, bitter orange, etc. It is a very important bioactive ingredient in citrus plants of the family rutaceae, and is considered to be vitamin P, also a biomarker for dried orange peel. The first part of Chinese pharmacopoeia 2020 edition specifies that the hesperidin content in dried orange peel is not lower than 3.5%. A great deal of researches show that the hesperidin not only has the functions of anti-inflammatory, anti-cancer, anti-oxidation and blood fat reduction, but also has potential therapeutic effects on a series of diseases such as cardiovascular and cerebrovascular diseases, nervous system diseases, mental diseases, cancers and the like. In addition, hesperidin is also widely applied to the industries of pharmacy, cosmetics and the like. Synephrine is distributed in the skin, flesh, juice, and seed of Citrus aurantium and its cultivar or Citrus sinensis, and is considered as a biomarker for citrus fruits, and synephrine content is also a quality index for evaluating commercial weight loss products. As a natural stimulant in the 21 st century, synephrine is often added to dietary supplements for exercise or fitness. More and more studies reveal its biological activities such as increasing blood pressure, anti-shock, promoting metabolism, increasing caloric consumption, increasing energy levels, oxidizing fat, losing weight, etc. To date, there has been no study on inhibition of ACR by hesperidin and synephrine.

Disclosure of Invention

The invention aims to: aiming at the problems existing in the prior art, the invention provides the application of SYN (SYN) or binary combination of Hesperidin (HES) and SYN (SYN) as the acrolein inhibitor, the SYN has good effect of inhibiting the acrolein, more importantly, the SYN can also play a role in binary combination of the hesperidin and the SYN, and when the binary combination is used, the trace SYN can be used as an accelerator or a catalyst to greatly improve the capturing activity of the hesperidin so as to synergistically inhibit the acrolein. The single synephrine or binary combination of synephrine and hesperidin disclosed by the invention can inhibit the acrolein existing in the environment and organisms, and solves the problems of poor thermal stability, low efficiency, side effect, potential safety hazard and the like of the existing acrolein inhibitor.

The invention also provides an environment and in-vivo acrolein inhibitor.

The technical scheme is as follows: in order to achieve the above purpose, the application of the synephrine or the combination of the synephrine and the hesperidin in the inhibition of acrolein is disclosed.

The invention relates to application of synephrine or combination of synephrine and hesperidin in preparation of an acrolein inhibitor.

Wherein, the structure of the hesperidin or the synephrine is as follows:

wherein, the synephrine or the combination of synephrine and hesperidin is used for inhibiting acrolein in the environment.

Wherein, the synephrine or the combination of synephrine and hesperidin is used for inhibiting acrolein in organisms.

Wherein, the synephrine and hesperidin are combined, and the dosage of the synephrine and hesperidin is 0.25 x IC ₅₀ -2*IC ₅₀ I.e. ICs each directed to acrolein ₅₀ The concentration is 0.25-2 times.

Preferably, the synephrine is combined with hesperidin in an amount of 2 x ic ₅₀ 。

Wherein the hesperidin or synephrine reduces the acrolein content by capturing acrolein to form an adduct product.

Wherein the addition products comprise an addition product HES-ACR of hesperidin and acrolein and a di-addition product SYN-2ACR of SYN-lin and acrolein, and the structures of the addition products are respectively shown as follows:

when the synephrine and the hesperidin are used in a binary combination mode, the synephrine is used as an accelerator or a catalyst to greatly improve the capture activity of the hesperidin, so that the effect of synergistically inhibiting the acrolein is achieved.

The invention relates to an environment and organism acrolein inhibitor, which is a preparation formed by taking synephrine as a unique component or binary combination or as a main component to be compounded and used together with other substances.

According to the invention, the hesperidin or the synephrine can be used as an acrolein inhibitor for the first time, and especially when the hesperidin and the synephrine are used in a binary combination mode, the inhibition activity and the reaction rate of the hesperidin can be greatly improved by using a trace amount of synephrine as an accelerator or by using the synephrine as a catalyst, and the reaction is very rapid, so that the acrolein is obviously and synergistically inhibited. Besides, hesperidin and synephrine are common dietary supplements, and have high safety as an acrolein inhibitor. In addition, hesperidin and synephrine are used as flavone and alkaloid which are most abundant in citrus fruits and plants such as lemon, orange, tangerine peel, hawksbill and the like, and are directly added or eaten, so that the limitation of exogenous addition can be avoided, and the effect of synergistically inhibiting acrolein can be better played in organisms.

The invention discovers that the synephrine can be used as an acrolein inhibitor, and simultaneously combines the synephrine with the hesperidin with low dosage, so that the capturing activity of the hesperidin on the acrolein can be obviously improved, and the hesperidin is promoted to capture more acrolein, thereby achieving the effect of synergistically inhibiting the acrolein. Hesperidin is the most abundant flavone in citrus, but the capturing activity of hesperidin is not high, but the research of the invention shows that the capturing performance of hesperidin can be greatly improved by adding a little synephrine. Compared with single-use hesperidin, the inhibition effect of hesperidin on ACR is obviously improved after the synephrine is added in a trace amount, the generation amount of HES-ACR which is a first adduct product of hesperidin and acrolein is about 3 times that of hesperidin when single-use hesperidin is adopted, and the SYN-2ACR content of the synephrine and acrolein is basically unchanged compared with that of the synephrine which is single-use hesperidin, so that the synephrine fully plays a role of an accelerator or a catalyst in binary combination. Meanwhile, the two have good inhibition effects under the conditions of low temperature and high temperature.

The beneficial effects are that: compared with the prior art, the invention has the following advantages:

the invention provides the inhibition effect of the hesperidin or the synephrine on the acrolein for the first time, particularly when the hesperidin and the synephrine are used in a binary combination mode, the synephrine is used as an accelerator or a catalyst to synergistically inhibit the acrolein with the hesperidin, and the content of the acrolein can be reduced efficiently. The hesperidin and the synephrine are taken as the acrolein inhibitor, can directly play roles in natural forms such as dried orange peel, orange and the like, remove acrolein in the environment and organisms, avoid covalent bonding with nucleophilic sites of biological macromolecules such as DNA, protein, nucleotide and the like, further avoid forming various irreversible harmful addition or crosslinking products, and prevent the damage to human bodies. The binary combination of the hesperidin and the synephrine is used as the acrolein inhibitor, so that the generation amount and the generation speed of HES-ACR which are an addition product of the hesperidin and the acrolein can be obviously improved, and the inhibition activity and the inhibition effect of the acrolein are obviously improved; the problems of poor heat stability, low efficiency, side effect and potential safety hazard of the synthetic inhibitor of part of inhibitors are avoided.

Drawings

FIG. 1 is a mass spectrum of hesperidin and its adduct with ACR;

FIG. 2 is a mass spectrum of synephrine and its adduct with ACR;

FIG. 3 is a graph depicting the inhibition of ACR by hesperidin or synephrine under simulated processing conditions;

FIG. 4 is a graph simulating the inhibition of ACR by hesperidin or synephrine under in vivo conditions;

FIG. 5 is an IC of hesperidin and synephrine under simulated processing conditions ₅₀ A value;

FIG. 6 is a graph of inhibition of ACR and Fa-CI for binary combination of hesperidin and synephrine under simulated processing conditions;

fig. 7 shows the mechanism of synergistic inhibition of ACR by hesperidin and synephrine when incubated for different times in binary combination.

Detailed Description

The invention will be further illustrated with reference to examples.

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

Example 1

Purification and structural investigation of the adduct of hesperidin or synephrine with ACR

(1) Experimental materials and instruments

Hesperidin (97%, HPLC) and synephrine (98%, HPLC) (Shanghai source leaf biotechnology limited); acrolein (ACR, 98% aqueous solution, analytically pure, eastern western chemical industry limited); methanol (analytically pure, shanghai national pharmaceutical Congress chemical reagent Co., ltd.); ethyl acetate (analytically pure, shanghai national pharmaceutical Congress chemical reagent Co., ltd.); purified water (Hangzhou Waha group Co., ltd.).

AVANCE 400MHz nuclear magnetic resonance apparatus (bruck); waters Xevo TQ-XS liquid Mass Spectrometry (Waters technologies, inc.).

(2) Experimental procedure

(1) Preparation of hesperidin and ACR adduct products

0.61g of hesperidin was weighed out and dissolved in 2.5mL of DMSO, and an ACR solution diluted with PBS (pH 7.0,0.1 mol/L) was added to react hesperidin with ACR at a molar ratio of 1:5 at 100℃for 0.5h. The product was then purified by reverse phase ODS column (2.6 cm. Times.30.0 cm), eluted with a 10% -30% methanol gradient, the 30% methanol eluate was collected, concentrated and then separated by silica gel column (3.0 cm. Times.30.0 cm), the eluate was collected (ethyl acetate: methanol=14:1, v/v), and lyophilized to give HES-ACR (158 mg), an adduct of hesperidin and ACR.

(2) Preparation of synephrine and ACR adducts

0.367g of synephrine is weighed and dissolved in 4mL of hot methanol, and ACR solution diluted with PBS (pH 7.0,0.1 mol/L) is added to react synephrine with ACR at a molar ratio of 1:10 for 0.5h at room temperature. Then, the reaction solution was concentrated and separated by a reversed-phase ODS column (2.6 cm. Times.30.0 cm), and eluted with a gradient of 2% -5% methanol, and 5% methanol eluate was collected, and lyophilized to give a SYN-2ACR (46 mg) which was a diadduct of SYN-lin and ACR.

(3) Structural identification of HES-ACR and SYN-2ACR

Dissolving HES-ACR in DMSO, dissolving SYN-2ACR in methanol to obtain 1mg/mL stock solution, respectively diluting with DMSO or methanol to 200ng/mL standard solution, and analyzing molecular weight by UPLC-MS/MS under the following specific conditions; 1D-NMR was used ¹ H， ¹³ C) Structural analysis was performed by 2D-NMR (HMQC, HMBC).

a) Chromatographic conditions

Instrument: waters Xevo TQ-XS

Chromatographic column: ACQUITY UPLC BEH C18 (2.1X105 mm, i.d.,1.7 μm)

Column temperature: 40 ℃; flow rate: 0.3mL/min; sample injection amount: 2 mu L

Mobile phase a:0.1% formic acid in water; mobile phase B: methanol

Elution gradient: 0-2min 5% B;2-3min 5-60% B;3-3.1min 60-95% B;3.1-4min 95% B;4-5min,95-5% B;5-6min,5% B.

b) Mass spectrometry conditions

Ion source: electrospray ion source (ESI); scanning mode: scanning positive ions; detection mode: multiple Reaction Monitoring Scans (MRMs); capillary voltage: 1.5kv; desolventizing gas temperature: 500 ℃; desolventizing gas flow: 1000L/hr; taper hole air flow: 150L/hr; data acquisition and analysis software: massLynx 4.2. The main mass spectrum parameters are shown in table 1.

TABLE 1 Primary Mass Spectrometry parameters

(3) Experimental results

(1) HES-ACR structural identification

As shown in FIG. 1, the prepared HES-ACR has a parent ion mass of m/z 667[ M+H ] in positive ion mode as determined by LC-MS/MS] ⁺ The parent ion mass m/z 611[ M+H ] of the HES] ⁺ Many 56 (MW) _ACR =56), and its main fragment ion peak in MS/MS is m/z 359[ m+h ]] ⁺ Is formed by the loss of one molecule of glucose and rhamnose groups (m/z 308) from HES-ACR, which can be shown initially that HES-ACR is the adduct of one molecule of HES and ACR ¹ H NMR (400 Hz) and ¹³ c NMR (100 MHz) data are detailed in Table 2. As can be seen from Table 2, HES-ACR has a similar carbon skeleton from C-1 to C-10 as HES. First, the H-6 hydrogen signal in HES-ACR is lost, and 3 new hydrogen signals delta appear simultaneously _H 1.92、2.00(2H,m),δ _H 2.69, 2.76 (2H, m) and delta _H 4.75 (1H, m) and 3 new carbon signals delta appear _C 29.49、δ _C 15.80 and delta _C 97.02. Furthermore, HMBC profile results showed δ _H 1.92、2.00(H-11)、δ _H 4.75 (H-13) and delta respectively _C 169.01(C-7)、δ _C 101.13 (C-5) correlation. Due to HES-ACR ¹³ No carbonyl signal was found in the C NMR spectrum, and it was determined that the carbonyl group of ACR was bonded to the C-6 site of HES and formed a cyclic hemiacetal structure with the hydroxyl group at the C-5 site. Therefore, the HES-ACR structure is finally determined by combining the characteristics of the nuclear magnetic spectrum, and is a novel compound, and the structure is as follows:

TABLE 2 adduct of HES and HES-ACR ¹ H NMR (400 Hz) and ¹³ C NMR(100MHz)

(2) SYN-2ACR identification

As shown in FIG. 2, the prepared SYN-2ACR has a parent ion mass of m/z 262[ M+H ] in positive ion mode as determined by LC-MS/MS] ⁺ Mass m/z 168[ M+H ] parent ion to SYN] ⁺ In comparison, 2ACR molecules (m/z 56)) were added and 1H was lost ₂ O molecules (m/z 18). The secondary mass spectrum has fragment ion peak m/z 150[ M-112+H ]] ⁺ ，m/z 119[M-143+H] ⁺ ，m/z 91[M-171+H] ⁺ All are identical to the characteristic fragments of SYN. It is speculated that SYN-2ACR is the di-adduct of SYN and ACR reaction. SYN-2ACR ¹ H NMR (400 Hz) and ¹³ c NMR (100 MHz) data are detailed in Table 3.

As can be seen from Table 3, the NMR data from C-1 to C-6 of SYN and SYN-2ACR were compared, and SYN-2ACR had a benzene ring structure similar to SYN. 5 new hydrogen signals (delta) appear in SYN-2ACR _H 3.38s,2H；δ _H 9.58s,1H；δ _H 6.17d,1H；δ _H 6.54m,1H；δ _H 5.19dd, 2H) and 6 new carbon signals (. Delta. _C 48.28,δ _C 142.92,δ _C 194.00,δ _C 134.01,δ _C 131.58andδ _C 94.15 Presumably, two ACR molecules are adducted with SYN. HMBC pattern results show H-7 (delta) _H 3.38 With C-2' (delta) _C 57.93)、C-3′(δ _C 39.25 With the addition of ACR to the secondary amine groups of SYN. In addition, H-10 (delta) _H 6.17 Hydrogen signal with C-8 (delta) _C 142.92),C-9(δ _C 194.00 And C-11(δ _C 131.58 All related, indicating that the second ACR group is conjugated at the C-8 position. Therefore, the structure of SYN-2ACR is finally determined by combining the characteristics of the nuclear magnetic spectrum diagram, and the structure of the SYN-2ACR is a novel compound:

TABLE 3 adduct of SYN and SYN-2ACR ¹ H NMR (400 Hz) and ¹³ C NMR(100MHz)

example 2

ACR inhibitory Activity of hesperidin or synephrine

(1) Experimental materials and instruments

Hesperidin (97%, HPLC) and synephrine (98%, HPLC) (Shanghai source leaf biotechnology limited); 2, 4-dinitrophenylhydrazine (dnph·hcl, purity >98%, tokyo Chemical Industry); acrolein (ACR, 98% aqueous solution, analytically pure, eastern western chemical industry limited); acetonitrile (chromatographic purity, shanghai national pharmaceutical Congress chemical Co., ltd.); purified water (a company of the ouha group, hangzhou); sodium dihydrogen phosphate and disodium hydrogen phosphate are both analytically pure reagents (Shanghai national pharmaceutical Congress chemical reagent Co., ltd.).

Agilent Technologies 1260 high performance liquid chromatograph (Agilent, USA); ZQTY-70 bench vibration incubator (Shanghai know Chu instruments Co., ltd.); QL-861 vortex mixer (manufactured by Chemie Instrument Co., ltd., jiangsu sea gate).

(2) Experimental procedure

(1) Inhibition activity of hesperidin or synephrine on acrolein in water solution is carried out under simulated processing conditions

ACR solution was prepared with 0.1mol/L PBS at pH 7.0, HES solution was prepared with DMSO, and SYN solution was prepared with methanol. In a 2mL centrifuge tube, 0.8mL of ACR solution (final concentration of 0.5 mmol/L) and 0.8mL of HES solution (final concentration of 0.5-2.5 mmol/L) or SYN solution (final concentration of 0.25-2 mmol/L) are respectively added, equal volumes of DMSO or methanol solution are respectively used for replacing HES or SYN solution as blank control, and after vortex mixing, the hesperidin group and the synephrine group are respectively placed in a water bath kettle at 100 ℃ to react for 5, 15, 30, 60 minutes and 1, 5, 15 and 30 minutes in a dark place. And after the reaction is finished, taking 500 mu L of reaction solution, adding 300 mu L of DNPH solution, placing in a table shaking incubator at 37 ℃, carrying out light-proof derivatization at 220rpm for 2 hours, immediately carrying out ice bath after the completion, detecting the content of ACR-DNPH derivative by using high performance liquid chromatography, and calculating the inhibition rate of HES or SYN solution on ACR under the simulated processing condition. Three sets of replicates were made for each sample.

(2) The inhibition activity of hesperidin or synephrine on acrolein in water solution is simulated under in-vivo conditions

ACR solution was prepared with 0.1mol/L PBS at pH 7.4, HES solution was prepared with DMSO, and SYN solution was prepared with methanol. In a 2mL centrifuge tube, 0.8mL of ACR solution (final concentration of 0.5 mmol/L) and 0.8mL of HES solution (final concentration of 1-16 mmol/L) or SYN solution (final concentration of 0.5-2.5 mmol/L) are respectively added, equal volumes of DMSO or methanol solution are respectively used for replacing HES or SYN solution as blank control, and after vortex mixing, the hesperidin group and the synephrine group are respectively placed in a desk-top shaking incubator at 37 ℃ for light-proof reaction for 1, 2,4, 8, 16 hours and 1, 5, 15, 30 and 60 minutes. The derivatization and the determination method are the same as (1). Three sets of replicates were made for each sample.

(3) HPLC conditions

Chromatographic column: kromasil 100-5C18 chromatographic column (250 x 4.6mm i.d.,5 μm);

a detector: a DAD detector; column temperature: 30 ℃; detection wavelength: 372nm; sample injection amount: 20. Mu.L.

Elution conditions: mobile phase a: water (0.1% formic acid); mobile phase B: acetonitrile;

the elution was isocratic with 70% mobile phase B at a flow rate of 1.0mL/min for 8min.

(4) Calculation formula of ACR clearance rate

(3) Data analysis

Experimental data were analyzed by One-way analysis of variance (One-way Analysis of variance, ANOVA) using SPSS13.0 software and by statistics using Tukey's method, p <0.05 indicating significant differences. Wherein different capital letters represent significant differences (p < 0.05) at different reaction times at the same concentration; different lowercase letters represent significant differences (p < 0.05) at different concentrations at the same reaction time.

(4) Experimental results

It can be seen from fig. 3 and 4 that HES and SYN can effectively capture ACR in a time and dose dependent manner, whether in aqueous solution under simulated processing or in vivo conditions. After 15min incubation at 100deg.C, about 50% of the ACR was cleared by the addition of 2.0mmol/L HES, while more than 80% of the ACR was cleared by SYN. At 37 ℃, both the hesperidin and the synephrine show the inhibition activity on ACR, and provide a basis for clearing ACR in human bodies from the hesperidin and the synephrine. Furthermore, SYN showed better ACR capture capacity than HES at the same concentration and incubation time. There have been a number of studies demonstrating that flavonoids generally have a certain ACR capturing capacity, such as myricetin, phloretin, EGCG, etc. SYN, in turn, acts as an alkaloid, where it exhibits better reactivity than HES (a flavonoid), and is fast and efficient in inhibiting acrolein.

Example 3

ACR inhibition activity by binary combination of hesperidin and synephrine

(1) Experimental materials and instruments

(2) Experimental procedure

(1) Chou-Talalay combination method principle

The combination of two or more substances employs additive amounts in a constant ratio to approximate an IC of two or more substances ₅₀ The ratio (half-inhibitory concentration) is a ratio to approximate IC ₅₀ Values are midpoint, 0.125, 0.25, 0.5, 1, 2-fold midpoint dose combination. The combined inhibition of two or more substances can be assessed by Fa-CI (combination index) curves. CI (CI)<1，CI＝1，CI>1 respectively represent synergy, superposition and antagonism. Wherein in the synergistic effect, 0.1<CI<0.3 represents strong synergy, 0.3<CI<0.7 represents synergy, 0.7<CI<0.85 represents moderate synergy.

(2) Preparation of samples

Equal volumes of ACR (final concentration 0.5 mmol/L) were incubated with HES (final concentrations 0.5, 1, 2,4, 8 mmol/L) or SYN (final concentrations 0.125, 0.25, 0.5, 1, 2 mmol/L) at 100 ℃ for 30min, and equal amounts of DMSO or methanol were used as control samples to incubate for 30min. After the reaction was completed, 0.5mL of each sample was taken for the derivatization. After the derivatization, the content of ACR-DNPH derivatives was detected and all assays were repeated three times. IC for HES and SYN was calculated using CompuSyn software according to Chou-Talay rules ₅₀ Values. Then, at a final concentration of 0.125 x ic ₅₀ -2*IC ₅₀ The HES solution and SYN solution were incubated with ACR (final concentration 0.5 mmol/L) alone or in combination for 30min at 100deg.C. After the reaction was completed, 0.5mL of each sample was taken for the derivatization. The content of ACR-DNPH derivative was measured by high performance liquid chromatography in the same manner as in example 2, and all analyses were repeated three times.

(3) Experimental results

As can be taken from FIG. 5, HES and SYN IC ₅₀ Values 2.144 and 0.240mmol/L, respectively, further demonstrate that SYN exhibits better reactivity than HES, consistent with the results of the inhibition of ACR by both simulated processing conditions. According to FIG. 6, whether used alone or in combinationThe combination is binary, the HES and SYN have dose dependency on ACR inhibition, and the binary combination obviously eliminates more ACR than single use. The effect of the combined inhibition of two or more substances can be evaluated based on Fa-CI (combination index) curves, as can be seen from the Fa-CI curves of FIG. 6, when Fa>0.35 (inhibition ratio)>35%) CI<1, HES and SYN at 0.25 x IC ₅₀ -2*IC ₅₀ The binary combination in the range has synergistic effect on inhibiting ACR. When SYN and HES are combined in 2 x IC ₅₀ When used in binary combination, ci=0.30, which is shown to be a strong synergy, the ACR clearance or inhibition can reach 96%.

Example 4

Mechanism research of binary combined synergistic inhibition of ACR by hesperidin and synephrine

(1) Experimental materials and instruments

Hesperidin (97%, HPLC) and synephrine (98%, HPLC) (Shanghai source leaf biotechnology limited); HES-ACR (prepared in example 1); SYN-2ACR (prepared in example 1); acrolein (ACR, 98% aqueous solution, analytically pure, eastern western chemical industry limited); 2, 4-dinitrophenylhydrazine (dnph·hcl, purity >98%, tokyo Chemical Industry); methanol (chromatographic purity, shanghai national pharmaceutical Congress chemical Co., ltd.); purified water (a company of the ouha group, hangzhou); sodium dihydrogen phosphate and disodium hydrogen phosphate are both analytically pure reagents (Shanghai national pharmaceutical Congress chemical reagent Co., ltd.).

Xevo ^TM tQ-XS ultra-high performance liquid chromatography-mass spectrometry (Watertian technologies (Shanghai); ZQTY-70 bench vibration incubator (Shanghai know Chu instruments Co., ltd.); QL-861 vortex mixer (manufactured by Chemie Instrument Co., ltd., jiangsu sea gate).

(2) Experimental procedure

(1) Establishment of a Standard Curve

HES-ACR and SYN-2ACR are respectively dissolved in DMSO and methanol to prepare stock solution of 1mg/mL, and then are diluted with DMSO or methanol to prepare a series of standard solutions with concentration gradient of 0.010-0.750 mug/mL, and are filtered by an organic film of 0.22 mu m. mu.L of each sample was taken for UPLC-MS/MS detection, and the detection method was the same as in example 1.

(2) Synergistic inhibition of ACR by different additive amounts

According to the Chou-Talay algorithm, 0.5mLHES solution (final concentrations 150, 230, 330, 560, 1200 mg/L) and 0.5mL SYN solution (final concentrations 19, 29, 41, 70, 150 mg/L) were incubated with equal volumes of ACR (final concentrations 0.5 mmol/L) at 100deg.C for 30min, respectively, in single or dual use, with the same volumes of DMSO or methanol as the blank instead of the inhibitor. After the completion of the reaction, 20. Mu.L of acetic acid was added, followed by an ice bath immediately to terminate the reaction. 0.5mL of each sample was subjected to the derivatization described above. After dilution with methanol, filtration was performed with a 0.22 μm organic membrane. mu.L of each was analyzed by LC-MS/MS to determine the amount of HES and SYN adducts with ACR. The assay was performed as in example 1 and all assays were repeated three times.

(3) Synergistic inhibition of ACR by different incubation times

0.5mL HES solution (final concentration 330 mg/L) and 0.5mL SYN solution (final concentration 41 mg/L) were incubated with equal volumes of ACR (final concentration 0.5 mmol/L) at 100deg.C for 30min, respectively, and the same volumes of DMSO or methanol were used as blanks instead of inhibitors. After the completion of the reaction, 20. Mu.L of acetic acid was added, followed by an ice bath immediately to terminate the reaction. After dilution with methanol, filtration was performed with a 0.22 μm organic membrane. mu.L of each sample was taken for LC-MS/MS detection. The assay was performed as in example 1 and all assays were repeated three times.

(3) Experimental results

The standard curve for HES-ACR is y=685.956x+5455.4, r ² Standard curve for syn-2ACR of y=42357.1x+916394, r=0.998 ² =0.993. The detection limits (LODs, S/n=3) for HES-ACR and SYN-2ACR were 0.4 and 0.6ng/mL, respectively, and the quantification limits (LOQs, S/n=10) were 1.2 and 1.8ng/mL, respectively.

It can be seen from table 4 that, whether HES and SYN are used singly or in combination, both the inhibition of ACR and the adduct content increase significantly with increasing additive amounts, again demonstrating that ACR inhibition is positively correlated with adduct formation. In addition, compared with HES alone, the inhibition effect of HES on ACR after SYN addition is obviously improved, the generation amount of HES-ACR is about 3 times that of HES alone, and the SYN-2ACR content is basically unchanged compared with SNY alone, so that the effect of a tiny amount of SYN serving as a promoter or a catalyst in binary combination is fully shown.

TABLE 4 synergistic inhibition mechanism of ACR by binary combination of HES and SYN at different concentrations

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On the other hand, the variation of the adduct content over time was investigated when HES and SYN were used in binary combination at fixed doses. As shown in FIG. 7, SYN exerts its maximum ability to capture ACR at 1min of incubation, when SYN-2ACR content is comparable to the adduct content of SYN alone for 30min. The SYN-2ACR content was essentially unchanged with prolonged incubation time, while the HES-ACR content increased sharply, much higher than the adduct content of HES alone. This demonstrates from another perspective that SYN is a HES promoter or catalyst. The action mechanism of SYN as HES promoter or catalyst is effectively analyzed, namely, the activity and the capturing speed of capturing acrolein of HES are obviously improved, and HES captures more ACRs to form more addition products, so that the effect of synergistically inhibiting the ACRs is achieved. HES is the most abundant flavone in citrus, but its capture activity for ACR is not high, but its capture performance can be greatly improved by adding a small amount of SYN in the present invention.

Claims

1. Use of synephrine in combination with hesperidin for inhibiting acrolein; the synephrine and hesperidin form an addition product by capturing acrolein, so that the acrolein content is reduced; the adduct comprises an adduct HES-ACR of hesperidin and acrolein and an adduct SYN-2ACR of SYN-acrolein, and the structures of the adducts are respectively shown as follows:

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the synephrine and hesperidin are combined, and the using amount of the synephrine and hesperidin is IC (integrated circuit) for acrolein respectively ₅₀ 0.25-2 times of concentration; when the synephrine and the hesperidin are used in a binary combination mode, the synephrine is used as an accelerator or a catalyst to improve the capture activity of the hesperidin, so that the effect of synergistically inhibiting the acrolein is achieved.

2. The use of synephrine in combination with hesperidin for the preparation of an acrolein inhibitor; the synephrine and hesperidin form an addition product by capturing acrolein, so that the acrolein content is reduced; the adduct comprises an adduct HES-ACR of hesperidin and acrolein and an adduct SYN-2ACR of SYN-acrolein, and the structures of the adducts are respectively shown as follows:

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3. The use according to claim 1 or 2, characterized in that the use of synephrine in combination with hesperidin for inhibiting acrolein in an environment.

4. The use according to claim 1 or 2, characterized in that the synephrine is used in combination with hesperidin for inhibiting acrolein in an organism.

5. An environmental and biological acrolein inhibitor, characterized in that the inhibitor is formed into a preparation by binary combination of synephrine and hesperidin or compound and co-use with other substances as main components; the binary combination of synephrine and hesperidin reduces the content of acrolein by capturing the acrolein to form an addition product; the adduct comprises an adduct HES-ACR of hesperidin and acrolein and an adduct SYN-2ACR of SYN-acrolein, and the structures of the adducts are respectively shown as follows:

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