A preparation method of chloroquinone

Abstract

The present invention discloses a method for preparing chloroquine. Polyphenol oxidase is added to a chlorogenic acid solution, oxygen is introduced, and chloroquine is prepared after mixed oxidation under water bath conditions of 25 to 38°C. The chloroquine is quenched with a nucleophilic reagent sodium benzenesulfinate to form a stable binding product. The working curve is drawn by ultra-high performance liquid chromatography-mass spectrometry, and the binding product is qualitatively and quantitatively detected to replace the detection of quinone substances. The purity of the prepared sodium benzenesulfinate of chloroquine reaches more than 95%, which can be used for quantitative determination. In addition, the preparation method of chloroquine in the present invention is low in cost, simple and convenient, easy to operate, and is conducive to popularization and use.

Description

Technical Field

[0001] The invention belongs to the technical field of preparation of quinone compounds, and particularly relates to a method for preparing chloroquinone. Background Art

[0002] Chlorogenic acid (CGA), also known as 5-O-caffeoylquinic acid, is a dehydrated acid produced by caffeic acid and quinic acid. It is produced in the shikimic acid pathway of plants during aerobic respiration. CGA is widely present in plants and is the main polyphenol in fruits and vegetables. There is a large amount of CGA in potatoes, accounting for more than 90% of its total phenolic compounds. Potatoes are prone to browning after being cut. During the browning process, due to the damage of plant tissues, the phenolic compounds and polyphenol oxidase in them are exposed to oxygen, which triggers the oxidation of phenolic substances into quinones. CGA is the best substrate for polyphenol oxidase. It is easily catalyzed into semiquinone in the presence of oxygen, and semiquinone disproportionately forms the corresponding orthoquinone, namely chlorogenicquinone (CGAQ). Orthoquinone substances are key intermediates with high reactivity. Once formed, they will rapidly undergo nucleophilic addition or redox reactions. Sodium benzenesulfinate can be used as a nucleophilic reagent to quench quinone, prevent it from continuing to react, and form a stable binding product. Chlorogenic acid is an oxidized product of chlorogenic acid. The two have similar structures and are derivatives of each other. Chlorogenic acid substances have multiple effects such as antioxidant, antibacterial, antiviral, anti-tumor, hypoglycemic, hypolipidemic, and hypotensive, and are widely used in food, medicine and other fields.

[0003] At present, the preparation of chloroquine mainly includes three methods: one is to extract it from fruits and vegetables, the second is to oxidize it with periodic acid resin, and the third is to obtain it through electrochemical methods. The extraction and purification process from fruits and vegetables is complicated and the yield is not high; the oxidation site is not easy to control with periodic acid resin oxidation, and by-products are easily obtained; the electrochemical method has complex instruments and is not easy to obtain and operate. Compared with the first three methods, the yield of chloroquine prepared by oxidizing chlorogenic acid with polyphenol oxidase reaches more than 80%, and the purity of chloroquine sodium benzenesulfinate reaches more than 95%, which can be used for quantification, and the preparation method is low-cost, simple and convenient, and easy to operate, which can provide a reference for the research of chloroquine and the rational development and utilization of resources. Summary of the invention

[0004] Technical problems to be solved: In view of the above technical problems, the purpose of the present invention is to provide a method for preparing chloroquinone, by using polyphenol oxidase to oxidize chlorogenic acid to prepare chloroquinone, using a nucleophilic reagent sodium benzenesulfinate to quench chloroquinone, overcoming the shortcomings of quinone substances such as instability, low oxidation yield, and easy oxidative polymerization, so that a stable binding product is formed, and the detection of the binding product is replaced by the detection of the quinone substances. The purity of the prepared sodium benzenesulfinate of chloroquinone reaches more than 95%, which can be used for quantitative determination. The preparation method of the present invention is low in cost, simple and convenient, easy to operate, and the maximum yield of chloroquinone can reach more than 80%.

[0005] Technical solution: A method for preparing chloroquinone, comprising the following steps:

[0006] S1. Weigh chlorogenic acid and dissolve it in phosphate buffer solution to obtain a 1.5-2.5 mmol / L chlorogenic acid solution;

[0007] S2. Weigh polyphenol oxidase and dissolve it in the chlorogenic acid solution to obtain a mixed solution;

[0008] S3. introducing oxygen into the mixed solution and carrying out oxidation reaction under water bath conditions of 25 to 38°C;

[0009] S4. Take 10 μL of the solution after the reaction, add 990 μL of sodium benzenesulfinate solution, mix well, and prepare chloroquinone sodium benzenesulfinate. After passing through a 0.22 μm nylon organic filter, inject it into a brown sample bottle for UPLC-ESI-MS / MS detection.

[0010] Furthermore, the pH of the phosphate buffer solution in S1 is 5-7, and the preparation method is as follows: weigh a certain amount of NaH 2 PO 4 .H 2 O, dissolve in distilled water to 1000mL to obtain mother solution A (100-400mmol / L); weigh a certain amount of Na 2 HPO 4 .2H 2 O, dissolve in distilled water to 1000mL to obtain mother solution B (100-400mmol / L); mix mother solution A and mother solution B, and adjust the pH to 5-7.

[0011] Furthermore, the mass ratio of polyphenol oxidase to chlorogenic acid is 850:71.

[0012] Furthermore, the activity of the polyphenol oxidase is 1000U / mL to 2000U / mL.

[0013] Furthermore, the flow rate of oxygen in S3 is 1 L / min, the concentration is 90-99%, and the oxidation reaction time is 20-90 s.

[0014] Furthermore, the concentration of the sodium benzenesulfinate solution in S4 is 0.04 mmol / L, and the solvent used is 80% ethanol. Sodium benzenesulfinate is a nucleophilic reagent, which reacts rapidly with quinone to form a corresponding adduct, and the dicarbonyl structure of quinone disappears, thereby preventing further reaction of quinone.

[0015] Furthermore, the UPLC-ESI-MS / MS detection uses a liquid chromatography-mass spectrometer Waters Acquity UPLC system, and the detection conditions are as follows:

[0016] (1) Liquid chromatography conditions:

[0017] Chromatographic column: ACQUITY UPLC@HSS T3 column, 2.1 mm × 100 mm, 1.8 μm;

[0018] Mobile phase: A: 0.1% formic acid in water, B: methanol;

[0019] Elution method: gradient elution;

[0020] Liquid phase gradient program: 0.00-1.00 min, 99% A; 1.00-6.00 min, 99%-0% A; 6.01-7.10 min, 99% A; column temperature: 40°C;

[0021] Sample chamber temperature: 10°C;

[0022] Injection volume: 3 μL;

[0023] Flow rate: 0.3 mL / min;

[0024] (2) Mass spectrometry conditions:

[0025] Mass spectrometer: triple quadrupole mass spectrometer;

[0026] Ion source: electrospray ion source;

[0027] Capillary voltage: 3.0kV;

[0028] Desolvation gas-nitrogen temperature: 350℃;

[0029] Desolvation gas flow rate: 650L / Hr

[0030] Cone hole gas flow: 150L / h;

[0031] Scanning mode: multiple reaction detection mode;

[0032] Data were collected by screening parent and daughter ions simultaneously in MRM mode. All the above operations were controlled by MassLynx software. Mass spectrometry (MRM) parameters:

[0033]

[0034] Table Note: * indicates quantitative ion

[0035] Further, the UPLC-ESI-MS / MS detection comprises the following steps:

[0036] (1) Preparation of standard working curve: Prepare sodium chloroquinone benzenesulfinate standard solution with 80% ethanol solution, then dilute the standard solution with 80% ethanol into six concentration gradients, and filter through a 0.22 μm nylon filter membrane;

[0037] (2) UPLC-ESI-MS / MS was used to analyze and count the peak areas of sodium chloroquinone benzenesulfinate at various concentrations. The standard working curve was drawn with the concentration of the standard solution as the abscissa and the chromatographic peak area of ​​the quantitative ion as the ordinate to obtain the linear regression equation and the goodness of fit R 2 .

[0038] Furthermore, the mobile phase A is prepared by taking 1.0 mL of chromatography-grade formic acid, adding ultrapure water to make the volume 1000 mL, that is, obtaining a 0.1% formic acid aqueous solution, and ultrasonically treating it for 10 minutes before use.

[0039] Further, the concentration gradient of the sodium chloroquinone benzene sulfinate standard solution is 50, 100, 500, 1000, 2000, 5000 μg / L. Further, the preparation method of the sodium chloroquinone benzene sulfinate standard solution is: use 80% ethanol solution (v / v) to prepare 1 mg / mL sodium chloroquinone benzene sulfinate solution reference solution as the mother solution. 100 μL of the mother solution is dispensed into a 1 mL centrifuge tube, the ethanol is removed by nitrogen blowing, and the tube is freeze-dried and stored at -20°C. When preparing the standard curve, take a 1 mL centrifuge tube containing freeze-dried powder of sodium chloroquinone benzenesulfinate solution, add 1 mL of 80% ethanol (v / v) to dissolve it, and dilute it with 80% ethanol (v / v) to 50, 100, 500, 1000, 2000, and 5000 μg / L chloroquinone solution standard solutions in sequence, pass through a 0.22 μm nylon organic filter, and inject it into a brown sample bottle for UPLC-ESI-MS / MS detection.

[0040] Furthermore, the goodness of fit R 2 At least 0.99.

[0041] Beneficial effects: Compared with the prior art, the present invention has the following advantages:

[0042] 1. The present invention utilizes polyphenol oxidase to oxidize chlorogenic acid to prepare chloroquine. The preparation method thereof is low in cost, simple and convenient, easy to operate, and the maximum yield of chloroquine can reach more than 80%. The purity of the prepared chloroquine sodium benzenesulfinate solution reaches more than 95%, which can be used for quantitative determination.

[0043] 2. The present invention overcomes the disadvantages of quinone substances being unstable and prone to oxidative polymerization, and uses a nucleophilic reagent, sodium benzenesulfinate, to quench chloroquinone to form a stable binding product, and the detection of the binding product replaces the detection of quinone substances.

[0044] 3. In the preparation method of chloroquine provided by the present invention, UPLC-ESI-MS / MS is used to quantitatively analyze the chloroquine solution, which has the advantages of high sensitivity, short analysis time and accurate quantification, and can be applied to the fields of medicine and food. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] The accompanying drawings are used to provide a further understanding of the present invention and constitute a part of the specification. Together with the embodiments of the present invention, they are used to explain the present invention and do not constitute a limitation of the present invention. Figure 1 It is the main chemical reaction formula of the present invention. DETAILED DESCRIPTION

[0046] The technical solution of the present invention will be clearly and completely described below in combination with the embodiments of the present invention and the accompanying drawings. Obviously, the described embodiments are not limitations of the present invention.

[0047] Example 1

[0048] A method for preparing chloroquine comprises the following steps:

[0049] S1. Weigh 1.42 mg of chlorogenic acid and dissolve it in 2 mL of phosphate buffer solution (pH = 5.8) to obtain a 2 mmol / L chlorogenic acid solution;

[0050] S2. Weigh 17.0 mg of polyphenol oxidase with a concentration of 2000 U / mL and dissolve it in the chlorogenic acid solution to obtain a mixed solution;

[0051] S3. Introduce oxygen into the mixed solution at a flow rate of 1 L / min and a concentration of 99%, and carry out oxidation reaction in a 35°C water bath for 60 seconds; S4. Take 10 μL of the solution after the reaction, add 990 μL of sodium benzenesulfinate (0.04 mmol / L) solution, mix well, and prepare chloroquinone sodium benzenesulfinate. After passing through a 0.22 μm nylon organic filter, inject into a brown sample bottle for UPLC-ESI-MS / MS detection.

[0052] Example 2

[0053] A method for preparing chloroquine comprises the following steps:

[0054] S1. Weigh 1.42 mg of chlorogenic acid and dissolve it in 2 mL of phosphate buffer solution (pH = 5.8) to obtain a 2 mmol / L chlorogenic acid solution;

[0055] S2. Weigh 17.0 mg of polyphenol oxidase with a concentration of 1000 U / mL and dissolve it in the chlorogenic acid solution to obtain a mixed solution;

[0056] S3. Introduce oxygen into the mixed solution at a flow rate of 1 L / min and a concentration of 99%, and carry out oxidation reaction in a 35°C water bath for 90 seconds; S4. Take 10 μL of the solution after the reaction, add 990 μL of sodium benzenesulfinate (0.04 mmol / L) solution, mix well, and prepare chloroquinone sodium benzenesulfinate. After passing through a 0.22 μm nylon organic filter, inject into a brown sample bottle for UPLC-ESI-MS / MS detection.

[0057] Example 3

[0058] A method for preparing chloroquine comprises the following steps:

[0059] S1. Weigh 1.42 mg of chlorogenic acid and dissolve it in 2 mL of phosphate buffer solution (pH = 6.5) to obtain a 2 mmol / L chlorogenic acid solution;

[0060] S2. Weigh 17.0 mg of polyphenol oxidase with a concentration of 2000 U / mL and dissolve it in the chlorogenic acid solution to obtain a mixed solution;

[0061] S3. Introduce oxygen into the mixed solution at a flow rate of 1 L / min and a concentration of 99%, and carry out oxidation reaction in a 25°C water bath for 20 seconds; S4. Take 10 μL of the solution after the reaction, add 990 μL of sodium benzenesulfinate (0.04 mmol / L) solution, mix well, and prepare chloroquinone sodium benzenesulfinate. After passing through a 0.22 μm nylon organic filter, inject into a brown sample bottle for UPLC-ESI-MS / MS detection.

[0062] Comparative Example 1

[0063] A method for preparing chloroquine comprises the following steps:

[0064] S1. Weigh 1.42 mg of chlorogenic acid and dissolve it in 2 mL of phosphate buffer solution (pH = 5.8) to obtain a 2 mmol / L chlorogenic acid solution;

[0065] S2. Pass oxygen into the chlorogenic acid solution at a flow rate of 1 L / min and a concentration of 99%, and carry out oxidation reaction in a 35°C water bath for 60 seconds; S3. Take 10 μL of the solution after the reaction, add 990 μL of sodium benzenesulfinate (0.04 mmol / L) solution, mix well, and prepare sodium chloroquinone benzenesulfinate. After passing through a 0.22 μm nylon organic filter, inject into a brown sample bottle for UPLC-ESI-MS / MS detection.

[0066] Test Results

[0067] The conversion rate and purity of the sodium chloroquinone benzenesulfinate samples prepared in Examples 1 to 3 and Comparative Example 1 were tested, and the sodium chloroquinone benzenesulfinate was analyzed using UPLC-ESI-MS / MS:

[0068] (1) Liquid chromatography conditions:

[0069] Chromatographic column: ACQUITY UPLC@HSS T3 column, 2.1 mm × 100 mm, 1.8 μm;

[0070] Mobile phase: A: 0.1% formic acid in water, B: methanol;

[0071] Elution method: gradient elution;

[0072] The liquid phase gradient program was: 0.00-1.00 min, 99% A; 1.00-6.00 min, 99%-0% A; 6.01-7.10 min, 99% A; column temperature: 40°C;

[0073] Sample chamber temperature: 10°C;

[0074] Injection volume: 3 μL;

[0075] Flow rate: 0.3 mL / min;

[0076] (2) Mass spectrometry conditions:

[0077] Mass spectrometer: triple quadrupole mass spectrometer;

[0078] Ion source: electrospray ion source;

[0079] Capillary voltage: 3.0kV;

[0080] Desolvation gas-nitrogen temperature: 350℃;

[0081] Desolvation gas flow rate: 650L / Hr

[0082] Cone hole gas flow: 150L / h;

[0083] Scanning mode: multiple reaction detection mode;

[0084] Data were collected by screening parent and daughter ions simultaneously in MRM mode. All the above operations were controlled by MassLynx software. Mass spectrometry (MRM) parameters:

[0085]

[0086] Note: * indicates quantitative ion.

[0087] Preparation of standard working curve: Use 80% ethanol solution (v / v) to prepare 1mg / mL sodium chloroquinone benzenesulfinate reference solution as the mother solution. Dispense 100μL of the mother solution into 1mL centrifuge tubes, remove methanol with nitrogen blow, freeze-dry and store at -20℃. When making the standard curve, take a 1mL centrifuge tube containing lyophilized powder of sodium chloroquinone benzenesulfinate, add 1mL 80% ethanol (v / v) to dissolve, and dilute with 80% ethanol (v / v) to 50, 100, 500, 1000, 2000, 5000μg / L sodium chloroquinone benzenesulfinate standard solution in sequence, pass through a 0.22μm nylon organic filter and inject into a brown sample bottle for UPLC-ESI-MS / MS detection. UPLC-ESI-MS / MS was used to perform detection and analysis according to the above chromatography-mass spectrometry conditions, and the peak area of ​​sodium chloroquinone benzenesulfinate at each concentration was counted. The standard working curve was drawn with the concentration of the standard solution as the abscissa and the chromatographic peak area of ​​the quantitative ion as the ordinate, and the linear regression equation was obtained as follows:

[0088] Y=148.5x–4703R 2 =0.9988

[0089] The test results are shown in Table 1.

[0090] Table 1 Conversion rate and purity of chloroquinone

[0091] Conversion rate (%) purity(%) Example 1 81.76 97.30 Example 2 56.32 95.87 Example 3 63.21 96.05 Comparative Example 1 0 0

[0092] As shown in Table 1, the conversion rate of the sodium chloroquinone benzene sulfinate prepared by the present invention is 56.32% to 81.76%, and the purity is 95.87% to 97.30%. In Comparative Example 1, no sodium chloroquinone benzene sulfinate was detected.

[0093] The above descriptions are merely embodiments of the present invention and are not intended to limit the protection scope of the present invention. Any equivalent structural transformations made using the contents of the present invention's specification and drawings, or directly or indirectly applied in other related technical fields, are included in the patent protection scope of the present invention.

Claims

1. A method for preparing chloroquine, characterized in that: The following steps are involved: S1. Weigh chlorogenic acid and dissolve it in a phosphate buffer solution to obtain a 1.5-2.5 mmol / L chlorogenic acid solution; the pH of the phosphate buffer solution is 5-7; S2. Weigh polyphenol oxidase and dissolve it in the chlorogenic acid solution to obtain a mixed solution; S3. oxygen is introduced into the mixed solution, and an oxidation reaction is carried out in a water bath at 25 to 38°C; the flow rate of oxygen in S3 is 1 L / min, the concentration is 90 to 99%, and the oxidation reaction time is 20 to 90 s; S4. Take 10 μL of the solution after the reaction, add 990 μL of sodium benzenesulfinate solution, mix well, and prepare chloroquinone sodium benzenesulfinate solution, wherein the concentration of the sodium benzenesulfinate solution is 0.04 mmol / L, and the solvent used is 80% ethanol; after passing through a 0.22 μm nylon organic filter, inject into a brown sample bottle for UPLC-ESI-MS / MS detection.

2. The method for preparing chloroquinone according to claim 1, characterized in that: The mass ratio of the polyphenol oxidase to chlorogenic acid is 850:

71.

3. The method for preparing chloroquinone according to claim 1, characterized in that: The activity of the polyphenol oxidase is 1000 U / mL to 2000 U / mL.

4. The method for preparing chloroquinone according to claim 1, characterized in that: The UPLC-ESI-MS / MS detection uses a liquid chromatography-mass spectrometer Waters Acquity UPLC system, and the detection conditions are as follows: (1) Liquid chromatography conditions: Chromatographic column: ACQUITY UPLC@HSS T3 column, 2.1 mm×100 mm, 1.8 μm; Mobile phase: A: 0.1% formic acid in water, B: methanol; Elution method: gradient elution; Liquid phase gradient program: 0.00-1.00 min, 99% A; 1.00-6.00 min, 99%-0% A; 6.01-7.10 min, 99% A; Column temperature: 40°C; Sample chamber temperature: 10°C; Injection volume: 3 μL; Flow rate: 0.3 mL / min; (2) Mass spectrometry conditions: Mass spectrometer: triple quadrupole mass spectrometer; Ion source: electrospray ion source; Capillary voltage: 3.0 kV; Desolvation gas-nitrogen temperature: 350°C; Desolvation gas flow rate: 650 L / Hr Cone hole gas flow: 150 L / h; Scanning mode: multiple reaction detection mode; Data were collected by screening parent / daughter ions simultaneously in MRM mode, all of which were controlled by MassLynx software; Mass spectrometry (MRM) parameters: ； Note: * indicates quantitative ion.

5. The method for preparing chloroquinone according to claim 1, characterized in that: The UPLC-ESI-MS / MS detection comprises the following steps: (1) Preparation of standard working curve: Prepare sodium chloroquinone benzenesulfinate standard solution with 80% ethanol solution, then dilute the standard solution with 80% ethanol into six concentration gradients, and filter through a 0.22 μm nylon filter membrane; (2) UPLC-ESI-MS / MS was used for analysis to count the peak areas of sodium chloroquinone benzenesulfinate at various concentrations. The standard working curve was drawn with the concentration of the standard solution as the horizontal axis and the chromatographic peak area of ​​the quantitative ion as the vertical axis to obtain the linear regression equation and the goodness of fit R. 2 .

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