A method for preparing a new hesperidin dihydrochalcone-propenal adduct

Abstract

The application discloses a preparation method of a new hesperidine dihydrochalcone-propylene aldehyde adduct, which comprises the following steps: adding new hesperidine dihydrochalcone into methanol to obtain mother liquor A; adding propylene aldehyde into methanol to obtain mother liquor B; mixing the mother liquor A and the mother liquor B, adding phosphate buffer solution, adjusting pH, heating and reacting, and filtering to obtain the new hesperidine dihydrochalcone-propylene aldehyde adduct. The new hesperidine dihydrochalcone-propylene aldehyde adduct prepared by the application can be used as a standard product of a propylene aldehyde reaction product in food detection, and the reaction system can generate the adduct within 5 min, and the clearance rate can reach 95.99% within 40 min. The new hesperidine dihydrochalcone-propylene aldehyde adduct can also be applied to qualitative determination of the propylene aldehyde adduct in rapid food detection. The preparation is simple, low in cost, efficient, free of consumption of a chromogenic agent, high in sample recovery rate, can effectively capture propylene aldehyde, and can control and effectively reduce the content of propylene aldehyde in food.

Description

Technical Field

[0001] This invention relates to the field of organic compound preparation, and more specifically, to a method for preparing a novel hesperidin dihydrochalcone-acrylaldehyde adduct. Background Technology

[0002] Acrolein is a highly reactive unsaturated aldehyde with sources that can be categorized as exogenous and endogenous. Exogenous acrolein is primarily produced through food processing, industrial emissions, organic combustion, and smoking, via lipid oxidation and Maillard reactions. Endogenous acrolein mainly originates from lipid peroxidation or polyamine oxidation within the human body. Acrolein is found in various foods, including fruits (0.01-0.05 ppm) and vegetables (up to 0.59 ppm). Studies have shown that long-term exposure to or consumption of foods rich in acrolein can have adverse effects on human health. Acrolein readily reacts with nucleophilic macromolecules in the body, interfering with DNA replication and transcription, leading to protein cross-linking, and potentially inducing chronic diseases such as lung cancer, cardiovascular disease, atherosclerosis, and diabetes. Current methods for controlling acrolein formation include reducing or eliminating precursor substances, altering processing methods and conditions, adding antioxidants, and adding scavengers such as amino acids and sulfur- and nitrogen-containing compounds.

[0003] CN 113149893 A discloses an acrolein-serine adduct and its preparation method. The method involves loading the sample onto a reverse-phase silica gel ODS resin, eluting with a 5% methanol aqueous solution, collecting the target adduct by a colorimetric method using 2,4-dinitrophenylhydrazine solution, verifying the purity by high-performance liquid chromatography, collecting the eluent, and freeze-drying to obtain the purified sample. This method has a high separation yield and an adduct purity of up to 99%. However, its drawbacks include the need for a colorimetric method and freeze-drying of the sample, resulting in a long reaction and preparation time, making it unsuitable for large-scale industrial production. The literature "Antioxidant and Acrolein-Acrolein Adduct Activity" by Lu Yongling et al. studied the antioxidant and acrolein-scavenging activities of the adduct obtained by the reaction of myricetin and acrolein. They found that the adduct formed after myricetin captures acrolein still has strong antioxidant and acrolein scavenging abilities, and can reduce the content of acrolein in the system. Myricetin is a flavonol compound widely found in many natural plants such as myrica and has a variety of pharmacological activities. However, it has the disadvantages of being expensive and easily oxidized, making it difficult to apply to industrial production.

[0004] The existing technology involves loading the sample onto a reverse-phase silica gel ODS resin, eluting with a 5% methanol aqueous solution, collecting the target adduct using a 2,4-dinitrophenylhydrazine solution colorimetric method, verifying purity by high-performance liquid chromatography (HPLC), collecting the eluent, and freeze-drying to obtain the purified sample. This method achieves high separation yield and adduct purity up to 99%. However, it suffers from drawbacks such as the need for colorimetric methods and freeze-drying, resulting in long reaction and preparation times, making it unsuitable for industrial production. Furthermore, the raw materials are expensive, easily oxidized, and the complex preparation process further hinders its industrial application. Summary of the Invention

[0005] Therefore, to address the problem that existing technologies for preparing novel hesperidin dihydrochalcone-propenal adducts have long reaction times and are difficult to apply to industrial production, this invention provides a method for preparing novel hesperidin dihydrochalcone-propenal adducts, the specific technical solution of which is as follows:

[0006] A method for preparing a novel hesperidin dihydrochalcone-acrylaldehyde adduct includes the following steps:

[0007] Neohesperidin dihydrochalcone was added to methanol to obtain mother liquor A;

[0008] Acrolein was added to methanol to obtain mother liquor B;

[0009] The mother liquor A and the mother liquor B were mixed evenly, then phosphate buffer was added to adjust the pH, the reaction was heated, and after filtration, the neohesperidin dihydrochalcone-acrylaldehyde adduct was obtained.

[0010] Furthermore, in the mother liquor A, the ratio of neohesperidin dihydrochalcone to methanol is 0.05 g / mL to 0.08 g / mL.

[0011] Furthermore, in the mother liquor B, the addition ratio of acrolein to methanol is 1 μL: (10-15) μL.

[0012] Furthermore, according to the volume ratio, the ratio of mother liquor A to mother liquor B is (2-5):1.

[0013] Further, adjust the pH to 7.4.

[0014] Furthermore, the heating reaction is carried out at a temperature of 35°C to 45°C for a duration of 25 min to 60 min.

[0015] Furthermore, the heating reaction is carried out at a temperature of 40°C for a duration of 40 minutes.

[0016] Furthermore, the heating reaction is carried out under light-proof oscillator conditions, and the oscillator speed is 200 r / min to 300 r / min.

[0017] Furthermore, the filtration process employs an organic microporous membrane with a flow rate of 0.45 μL to 0.60 μL.

[0018] Furthermore, the molecular structural formula of the neohesperidin dihydrochalcone-acrylaldehyde adduct is as follows:

[0019]

[0020] The novel hesperidin dihydrochalcone-acrylaldehyde adduct prepared in the above scheme can be used in the field of food detection as a standard for acrolein reaction products in food testing. The adduct is generated within 5 minutes, and the clearance rate reaches 95.99% within 40 minutes. It can also be applied to the qualitative analysis of acrolein adducts in rapid food testing. The preparation method of the novel hesperidin dihydrochalcone-acrylaldehyde adduct of this invention is characterized by simple operation, low cost, high efficiency, no need for colorimetric reagents, and high sample recovery rate. Furthermore, in food additives, neohesperidin dihydrochalcone is used as a sweetener, with a sweetness 1500-1800 times that of the same mass of sucrose, and possesses antioxidant, hypoglycemic, hypolipidemic, hypocholesterol-lowering, low-calorie, non-toxic, and safe properties, making it suitable for the pharmaceutical, cosmetic, food, and feed industries. Furthermore, the preparation method described in this application can effectively capture acrolein, control and effectively reduce the content of acrolein in food, thus giving neohesperidin dihydrochalcone a broader prospect for development as a food additive to remove acrolein. Attached Figure Description

[0021] Figure 1 This is a schematic diagram of the liquid chromatography reaction of hesperidin dihydrochalcone with acrolein.

[0022] Figure 2 This is a schematic diagram of the reaction system of NHDC and ACR;

[0023] Figure 3 This is a schematic diagram illustrating the acrolein removal rate.

[0024] Figure 4 A schematic diagram of the nuclear magnetic resonance-1H spectrum of the reaction product of neohesperidin dihydrochalcone and acrolein (compound 1);

[0025] Figure 5 This is a schematic diagram of the primary mass spectrometry of the reaction product of neohesperidin dihydrochalcone and acrolein (compound 1). Detailed Implementation

[0026] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to its embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and do not limit the scope of protection of the invention.

[0027] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0028] A method for preparing a novel hesperidin dihydrochalcone-acrylaldehyde adduct according to one embodiment of the present invention includes the following steps:

[0029] Neohesperidin dihydrochalcone was added to methanol to obtain mother liquor A;

[0030] Acrolein was added to methanol to obtain mother liquor B;

[0031] The mother liquor A and the mother liquor B were mixed evenly, then phosphate buffer was added to adjust the pH, the reaction was heated, and after filtration, the neohesperidin dihydrochalcone-acrylaldehyde adduct was obtained.

[0032] In one embodiment, the ratio of neohesperidin dihydrochalcone to methanol in the mother liquor A is 0.05 g / mL to 0.08 g / mL.

[0033] In one embodiment, the ratio of acrolein to methanol in the mother liquor B is 1 μL: (10-15) μL.

[0034] In one embodiment, the ratio of mother liquor A to mother liquor B by volume is (2-5):1.

[0035] In one embodiment, the pH is adjusted to 7.4.

[0036] In one embodiment, the heating reaction is carried out at a temperature of 35°C to 45°C for a duration of 25 min to 60 min.

[0037] In one embodiment, the heating reaction is carried out at a temperature of 40°C for a duration of 40 minutes.

[0038] In one embodiment, the heating reaction is carried out under light-shielded oscillator conditions, wherein the oscillator rotates at a speed of 200 r / min to 300 r / min.

[0039] In one embodiment, the filtration process uses an organic microporous membrane with a flow rate of 0.45 μL to 0.60 μL.

[0040] In one embodiment, the molecular structure of the neohesperidin dihydrochalcone-acrylaldehyde adduct is as follows:

[0041]

[0042] The novel hesperidin dihydrochalcone-acrylaldehyde adduct prepared in the above scheme can be used in the field of food detection as a standard for acrolein reaction products in food testing. The adduct is generated within 5 minutes, and the clearance rate reaches 95.99% within 40 minutes. It can also be applied to the qualitative analysis of acrolein adducts in rapid food testing. The preparation method of the novel hesperidin dihydrochalcone-acrylaldehyde adduct of this invention is characterized by simple operation, low cost, high efficiency, no need for colorimetric reagents, and high sample recovery rate. Furthermore, in food additives, neohesperidin dihydrochalcone is used as a sweetener, with a sweetness 1500-1800 times that of the same mass of sucrose, and possesses antioxidant, hypoglycemic, hypolipidemic, hypocholesterol-lowering, low-calorie, non-toxic, and safe properties, making it suitable for the pharmaceutical, cosmetic, food, and feed industries. Furthermore, the preparation method described in this application can effectively capture acrolein, control and effectively reduce the content of acrolein in food, thus giving neohesperidin dihydrochalcone a broader prospect for development as a food additive to remove acrolein.

[0043] The implementation schemes of the present invention will now be described in detail with reference to specific embodiments.

[0044] Example 1:

[0045] A method for preparing a novel hesperidin dihydrochalcone-acrylaldehyde adduct includes the following steps:

[0046] Add 0.0613 g of neohesperidin dihydrochalcone to 1 mL of methanol to obtain mother liquor A;

[0047] 70.25 μL of acrolein was added to 710 μL of methanol to obtain mother liquor B;

[0048] Mix mother liquor A and mother liquor B in a volume ratio of 2:1 until homogeneous, then add 8 mL of phosphate buffer to adjust the pH to 7.4, seal, and heat at 40℃ and 200 r / min for 40 min. After dilution with chromatographic methanol, filter through a 0.45 μL organic microporous membrane to obtain the neohesperidin dihydrochalcone-acrylaldehyde adduct.

[0049] The content of neohesperidin dihydrochalcone-propenal adduct in the reaction system was determined by high performance liquid chromatography (HPLC). The HPLC chromatographic conditions were as follows: Caprisil C18-AQ column, 5 μm, 250 mm × 4.6 mm (pH 1.5-11); mobile phase A: 0.1% aqueous acetic acid; mobile phase B: acetonitrile (ACN); flow rate: 1 mL / min; injection volume: 10 μL; elution conditions: 30 min 10%-100% ACN. The results are as follows. Figure 1 As shown. From Figure 1 As can be seen, adducts are formed within 5 minutes of the reaction system, and the clearance rate can reach 95.99% within 40 minutes.

[0050] Example 2:

[0051] The M / mL acrolein-methanol solution is stock solution B (accurately pipette 351 μL acrolein + 4649 μL methanol).

[0052] ③ Prepare phosphate buffer (pH=7.4) as PBS.

[0053] 1. Prepare 800mL of distilled water in a suitable container.

[0054] 2. Add 8g of NaCl to the solution.

[0055] 3. Add 200 mg KCl to the solution.

[0056] 4. Add 1.44g of Na2HPO4 to the solution.

[0057] 5. Add 240 mg of KH2PO4 to the solution.

[0058] 6. Adjust the solution to the desired pH (usually pH≈7.4)

[0059] 7. Add distilled water until the volume is 1 liter.

[0060] ④ According to Table 1 below, add the mother liquor and required reagents into a 1.5 mL EP tube, seal the cap tightly with sealing film, and place it in a metal constant temperature shaker in a light-protected environment to carry out the reaction:

[0061] Parameters of the metal thermostatic oscillator: Rotation speed: 200 rpm; Reaction temperature: 40℃; Reaction time: 30 min.

[0062] Table 1:

[0063] Mother liquor A / μL Mother liquor B / μL MeOH:PBS MeOH / μL PBS / μL / 10 / 990 0 100 / / 900 0 100 10 9:1 790 100 100 10 8:2 690 200 100 10 7:3 590 300 100 10 6:4 490 400 100 10 5:5 390 500 100 10 4:6 290 600 100 10 3:7 190 700 100 10 2:8 90 800 100 10 1:9 0 890

[0064] i. Non-derivative: For each sample, pipette 100 μL into a 1.5 mL EP tube, add 10 μL of acetic acid, then add 890 μL of HPLC-grade methanol, and filter through a 0.45 μm microporous membrane. Then pipette another 100 μL of the filtered sample into a sample vial, add 400 μL of HPLC-grade methanol, and proceed with the analysis.

[0065] Injection volume: 5 μL

[0066] ii. Derivatization: Pipette 200 μL of each sample into a 1.5 mL EP tube, add 10 μL of acetic acid, then add 790 μL of saturated DNPH solution diluted 10 times, and place in a metal shaker for light-protected derivatization.

[0067] Parameters of metal thermostatic oscillator:

[0068] Speed: 200 rpm;

[0069] Reaction temperature: 40℃;

[0070] Reaction time: 60 min.

[0071] After derivatization, the sample was filtered through a 0.45 μm microporous membrane, and then 100 μL of the filtered sample was transferred to a sample vial and added to 400 μL of HPLC-grade methanol for analysis.

[0072] The HPLC chromatographic conditions were as follows: Caprisil C18-AQ column, 5 μm, 250 mm × 4.6 mm (pH 1.5-11); mobile phase A: 0.1% aqueous acetic acid; mobile phase B: acetonitrile (ACN); flow rate: 0.8 mL / min; injection volume: 5 μL; elution conditions: 0-12 min, 28-60% ACN; 12-16 min, 90% ACN; 16-20 min, 28% ACN. Figure 2 The reaction system of NHDC and ACR is composed of... Figure 2 It can be seen that in the reaction system of NHDC and ACR, the concentration of NHDC-A gradually increases with the increase of PBS ratio, and gradually stabilizes after 2:8, while the concentration of NHDC gradually decreases. The relationship between NHDC and NHDC-A is that of reactant consumption and product formation. Figure 3 The removal rate of acrolein, from Figure 3 As can be seen, the acrolein removal rate gradually increases with the increase of the PBS ratio, reaching a maximum of 96.35%.

[0073] Example 3:

[0074] A method for capturing acrolein includes the following steps:

[0075] Weigh 0.0613 g of neohesperidin dihydrochalcone, 2 mL of methanol, 8 mL of phosphate buffered saline (PBS, pH 7.4), and 74 μL of acrolein solution (70.25 μL, 95%, 0.1 mM / mL). Seal the container and react at 40 °C for 5 min, 10 min, 15 min, 20 min, 25 min, 30 min, 35 min, and 40 min. At each time point, take an appropriate amount of the reaction solution, dilute it with chromatographic methanol, filter it through a 0.45 μL organic microporous membrane, and determine the content of the adduct in the reaction system by HPLC.

[0076] Table 2 shows the capture results of acrolein in Example 3.

[0077]

[0078] As can be seen from Table 2, in the same reaction concentration system, the amount of acrolein removal rate increases with the increase of reaction time. When the reaction time is 35 min, the adduct capture rate is 96.35%, and the capture effect is obvious.

[0079] Example 4:

[0080] Isolation and purification of compounds

[0081] After the reaction in Example 1 was completed, the reaction solution was evaporated to dryness under reduced pressure (the HPLC chromatogram of reaction solution 1 is shown in [reference needed]). Figure 1 Add an appropriate amount of methanol to dissolve, filter to remove salt, concentrate the filtrate again under reduced pressure, collect the fraction using silica gel column chromatography and gel (LH-20) column chromatography, and evaporate to dryness using a rotary evaporator to obtain compound 1.

[0082] Structural characterization of compound 1:

[0083] A small amount of compound 1 was dissolved in deuterated methanol, and the NMR-1H spectrum of compound 1 was measured using an NMR spectrometer. The NMR-1H spectrum of compound 1 is shown below. Figure 4 The mass spectrum shows that the molecular ion peak of compound 1 is m / z = 668 (see details). Figure 5 ).

[0084] The 1H NMR (400 MHz, methanol-d4) values ​​for compound 1 are: δ 6.86 (d, J = 8.2 Hz, 1H), 6.79 (d, J = 2.2 Hz, 1H), 6.73 (dd, J = 8.1, 2.2 Hz, 1H), 6.27 (s, 1H), 5.64 (s, 1H), 5.41–5.37 (m, 1H), 5.26 (d, J = 7.3 Hz, 1H), 4.04–4.02 (m, 1H), 3.94–3.89 (m, 1H), 3.8 The following structures are obtained using the following parameters: 6(s,3H), 3.84–3.80(m,1H), 3.79–3.66(m,4H), 3.53–3.49(m,2H), 3.44(d,J=9.6Hz,1H), 3.42–3.36(m,2H), 2.90(q,J=9.8,8.6Hz,3H), 2.70(p,J=7.0,6.2Hz,1H), 2.04–1.72(m,2H), 1.22(d,J=6.0Hz,3H):

[0085]

[0086] The application of the neohesperidin dihydrochalcone-acrylaldehyde adduct in food detection is threefold: First, the reaction system is highly sensitive, with the adduct forming within 5 minutes, making it suitable for the qualitative analysis of acrolein adducts in rapid food testing. Second, it is the first time a sweetener has been reacted with acrolein; neohesperidin dihydrochalcone achieves a scavenging rate of up to 96.74% for acrolein. Given sufficient reaction time and substrate, it can largely eliminate acrolein generated during food processing. Third, as a food additive, neohesperidin dihydrochalcone is characterized by low calories, high sweetness, and excellent antioxidant properties. It is a natural antioxidant that can inhibit the formation of acrolein in food and can be used in various foods without being limited by reaction conditions. In food additives, neohesperidin dihydrochalcone is used as a sweetener, with a sweetness 1500-1800 times that of the same weight of sucrose. It also possesses characteristics such as lowering blood sugar, blood lipids, and cholesterol, low calories, non-toxicity, and safety, making it suitable for the pharmaceutical, cosmetic, food, and feed industries.

[0087] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0088] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. A method for preparing a novel hesperidin dihydrochalcone-acrylaldehyde adduct, characterized in that, The preparation method includes the following steps: Neohesperidin dihydrochalcone was added to methanol to obtain mother liquor A; Acrolein was added to methanol to obtain mother liquor B; The mother liquor A and the mother liquor B were mixed evenly, then phosphate buffer was added, the pH was adjusted, the reaction was heated, and after filtration, the neohesperidin dihydrochalcone-acrylaldehyde adduct was obtained. The molecular structure of the neohesperidin dihydrochalcone-propenal adduct is as follows: 。 2. The preparation method according to claim 1, characterized in that, In the mother liquor A, the ratio of neohesperidin dihydrochalcone to methanol is 0.05 g / mL to 0.08 g / mL.

3. The preparation method according to claim 1, characterized in that, In the mother liquor B, the ratio of acrolein to methanol is 1 μL: (10~15) μL.

4. The preparation method according to claim 1, characterized in that, According to the volume ratio, the ratio of mother liquor A to mother liquor B is (2~5):

1.

5. The preparation method according to claim 1, characterized in that, Adjust the pH to 7.

4.

6. The preparation method according to claim 1, characterized in that, The heating reaction is carried out at a temperature of 35℃ to 45℃ for a time of 25 min to 60 min.

7. The preparation method according to claim 6, characterized in that, The heating reaction was carried out at a temperature of 40°C for 40 minutes.

8. The preparation method according to claim 1, characterized in that, The heating reaction is carried out under light-proof oscillator conditions, and the oscillator speed is 200 r / min to 300 r / min.

9. The preparation method according to claim 1, characterized in that, The filtration process uses an organic microporous membrane with a flow rate of 0.45 μL to 0.60 μL.

Images