Detection method of mango urushiol

Abstract

The invention provides a method for detecting mango urushiol. The method comprises the following steps: (1) extracting; (2) purifying; and (3) detecting. The pretreatment bottleneck of a high-sugar and high-moisture sample is broken through a liquid nitrogen rapid freezing and crushing technology, and the problems of long drying pretreatment process time and urushiol loss in the drying process in traditional detection are solved; the improved betaine-lactic acid DES is used as an extracting agent to replace traditional trichloromethane, so that the extraction rate is increased, and green and efficient extraction is realized. In general, the invention provides a mango urushiol detection method with high sensitivity and operation safety.

Description

Technical field

[0001] The present invention relates to the technical field of component detection, and in particular to a method for detecting mango urushiol. [Background Technology]

[0002] Urushiol is a natural allergen secreted by mango flesh, peel, branches, and leaves. Contact with it can cause allergic reactions such as redness, swelling, and itching. Studies have shown that mango urushiol is structurally similar to poison ivy urushiol and belongs to the class of long-chain alkyl catechol compounds. Currently, urushiol testing is typically conducted according to national standard methods.

[0003] The current national standard method for testing has the following two major technical defects:

[0004] First, the national standard method uses dried raw materials as the test sample when detecting urushiol in raw materials. However, mangoes have a high moisture content of 70%-85%. Conventional hot air drying requires 48-72 hours, and freeze-drying takes even longer. Furthermore, during the drying process, urushiol undergoes structural isomerization due to high temperatures (>40°C) or oxidation. Furthermore, high-temperature dehydration of pectin forms a gel that encapsulates the urushiol, reducing extraction efficiency.

[0005] Second, the national standard method often uses chloroform as an organic solvent during the extraction and dissolution steps of urushiol. Chloroform is not only highly toxic and can cause irreversible damage to the liver and kidneys upon contact, but as a regulated organic solvent, its use and approval process are cumbersome, and its storage requires specialized explosion-proof cabinets with two people and double locks. This is costly and poses safety risks, which is inconsistent with the development trend of green chemistry.

[0006] Therefore, there is an urgent need to improve the existing mango urushiol detection method to overcome the above-mentioned defects. [Summary of the invention]

[0007] The present invention provides a method for detecting mango urushiol. The method breaks through the bottleneck of high-sugar and high-moisture sample pretreatment by using liquid nitrogen rapid freezing and crushing technology, solves the problems of long drying pretreatment time and urushiol loss during the drying process in traditional detection; improved betaine-lactic acid DES is used as an extraction agent to replace traditional chloroform, thereby improving the extraction rate and realizing green and efficient extraction.

[0008] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0009] A method for detecting mango urushiol, the method comprising the following steps:

[0010] (1) Extraction: Fresh mango tissue was crushed, added with a deep eutectic solvent, and ultrasonic-assisted extraction was performed to obtain an extract for later use;

[0011] (2) Purification: The extract was purified by a hydrophobic solid phase extraction column;

[0012] (3) Detection: The urushiol content was detected by high performance liquid chromatography (i.e., using the national standard method, standard number GB / T 41716-2022);

[0013] The low eutectic solvent is composed of a hydrogen bond acceptor and a hydrogen bond donor in a molar ratio of 1:1-3, the hydrogen bond acceptor is choline or betaine, and the hydrogen bond donor is lactic acid, capric acid or malic acid.

[0014] In the present invention, the fresh mango sample does not need to be dried, but is directly frozen and crushed by liquid nitrogen after sampling, with a crushing particle size of 80-100 mesh. It can be used immediately to avoid deterioration of urushiol due to exposure to oxidation.

[0015] In the present invention, further, the combination of hydrogen bond acceptor and hydrogen bond donor in the deep eutectic solvent is betaine-lactic acid, choline-decanoic acid or betaine-malic acid, and the molar ratio of the hydrogen bond acceptor to the hydrogen bond donor is 1:2.

[0016] In the present invention, further, the ultrasonic extraction conditions are: power 400-500W, temperature 30-40°C, time 20-30min, and the mass ratio of the extract to the sample is 10:1.

[0017] In the present invention, further, the extraction step adopts a step-by-step extraction method, first adding 50% volume of DES and shaking for 10 minutes, and then adding the remaining DES for ultrasonic treatment.

[0018] In the present invention, further, the purification step uses a C18 solid phase extraction column, the activation solvent is methanol, and the elution solvent is acetonitrile-water solution (70:30, v / v).

[0019] In the present invention, further, the high performance liquid chromatography GB / T 41716-2022 detection conditions include:

[0020] 1) Chromatographic column: C 18 , 250mm*4.6mm (inner diameter), particle size 5μm, or column efficiency equivalent;

[0021] 2) Mobile phase: acetonitrile-water (volume ratio 91:9);

[0022] 3) Injection volume: 5 μL;

[0023] 4) Flow rate: 0.60 mL / min;

[0024] 5) Detection wavelength: 277 nm;

[0025] 6) Column temperature: room temperature.

[0026] The present invention also provides a low eutectic solvent composition for extracting mango urushiol, which is prepared by mixing betaine and lactic acid in a molar ratio of 1:2, adding water to adjust the viscosity to 50-100 cP, and the pH value to 4.0-5.0.

[0027] The present invention also provides application of the above method in mango allergen screening, food quality and safety control, or cosmetic raw material detection.

[0028] The present invention also provides a mango urushiol detection kit, comprising the above-mentioned deep eutectic solvent composition for mango urushiol extraction, a standard urushiol reference substance and a solid phase extraction column.

[0029] In summary, due to the adoption of the above technical solution, the present invention has at least the following beneficial effects:

[0030] The present invention provides a method for detecting mango urushiol, which solves the problems existing in the prior art and is a method for detecting mango urushiol with high sensitivity and operational safety.

[0031] Specifically, the present application first uses fresh mango fruit as the test raw material, breaking through the traditional drying limitations and maintaining the activity of urushiol through liquid nitrogen freezing and crushing; avoiding the difficulty of drying mangoes, while preventing the influence of urushiol structural changes (such as urushiol dimerization) on the accuracy of the test results during the drying process, so that the test sample can more truly reflect the actual content of urushiol in mangoes, thereby improving the reliability of the test results;

[0032] In addition, this application has developed a low eutectic solvent for urushiol extraction. Betaine-lactic acid DES has both high extraction efficiency and green characteristics: (1) Lactic acid provides a weak acidic environment to stabilize the urushiol structure; (2) The quaternary ammonium cation of betaine forms a π-cation interaction with the benzene ring of urushiol; (3) The moderate viscosity is conducive to ultrasonic enhanced mass transfer. This system provides the first non-chlorinated solvent solution for urushiol detection. It is green and environmentally friendly, does not harm the health of operators, does not require complex regulatory procedures, simplifies the operating process, and reduces operational risks. Moreover, the low eutectic green extract has good solubility for mango urushiol and has a high recovery rate;

[0033] Finally, the present invention establishes an integrated detection platform to eliminate matrix interference (such as pectin and sugars). The method of the present invention not only solves the practical problems of existing methods, but also meets the requirements of green environmental protection and safe production, and has good practicality and promotion value. [Specific implementation method]

[0034] The following examples may help those skilled in the art to more fully understand the present invention, but shall not limit the present invention in any way.

[0035] Example 1: Preparation of deep eutectic solvent

[0036] This embodiment provides a method for preparing a deep eutectic solvent, which comprises the following steps:

[0037] Betaine (hydrogen bond acceptor) and lactic acid (hydrogen bond donor) are mixed in a molar ratio of 1:2, stirred at 60°C until a homogeneous transparent liquid is formed, and water is added to adjust the viscosity to 85 cP to obtain a deep eutectic solvent.

[0038] Example 2: Extraction of urushiol from fresh mango

[0039] The mango urushiol was extracted using the low eutectic solvent described in Example 1. Specifically, 10 g of Guifei mango pulp (maturity 80%) was taken, frozen with liquid nitrogen, and then ball-milled into powder (100 mesh). 100 mL of betaine-lactic acid DES was added, and ultrasonic treatment was performed at 40 ° C (450W, 25 min). The extraction step adopted a step-by-step extraction method, first adding 50% volume of DES and shaking for 10 minutes, then adding the remaining DES for ultrasonic treatment, centrifuging and taking the supernatant, and loading the supernatant onto a C18-SPE column (500 mg / 6 mL), eluting with 5 mL of water, 5 mL of 30% methanol, and 8 mL of acetonitrile-water (70:30) in sequence to obtain the mango urushiol.

[0040] Example 3: Detection of urushiol in fresh mangoes

[0041] (1) Sample preparation: 10 g of Guifei mango pulp (maturity 80%) was frozen in liquid nitrogen and then ball-milled into powder (100 mesh);

[0042] (2) DES extraction: 100 mL of betaine-lactic acid DES (the deep eutectic solvent described in Example 1) was added, ultrasonicated at 40°C (450W, 25 min), and the supernatant was collected by centrifugation;

[0043] (3) Purification: The supernatant was loaded onto a C18-SPE column (500 mg / 6 mL), washed sequentially with 5 mL of water, 5 mL of 30% methanol, and eluted with 8 mL of acetonitrile-water (70:30);

[0044] (4) High performance liquid chromatography (i.e., using the national standard method, standard number GB / T 41716-2022) analysis, conditions:

[0045] 1) Chromatographic column: C 18 , 250mm*4.6mm (inner diameter), particle size 5μm, or column efficiency equivalent;

[0046] 2) Mobile phase: acetonitrile-water (volume ratio 91:9);

[0047] 3) Injection volume: 5 μL;

[0048] 4) Flow rate: 0.60 mL / min;

[0049] 5) Detection wavelength: 277 nm;

[0050] 6) Column temperature: room temperature.

[0051] Example 4: Comparison of urushiol loss in different samples

[0052] This example tests the comparison of urushiol loss between fresh samples and dried samples;

[0053] Experimental design:

[0054] Ten groups of mango pulp samples (1 g each) were taken from the same mango, 5 groups were subjected to liquid nitrogen freeze-crushing using the method of the present application (present application group), and 5 groups were dried at 60°C to constant weight and then crushed (control group).

[0055] All samples were extracted with betaine-lactic acid DES and detected by high performance liquid chromatography (standard number GB / T 41716-2022).

[0056] Compare the effects of different sample selections on the content of mango urushiol, where mango urushiol includes saturated urushiol, monounsaturated urushiol, diunsaturated urushiol, and triunsaturated urushiol. The values recorded in the examples of this application are the sum of the above different types of urushiol (the same below);

[0057] The results are shown in Table 1:

[0058] Table 1

[0059] Sample type Measured urushiol content (μg / g) RSD (%) Difference rate compared with fresh group This application group 28.7±0.9 3.1 - control group 16.2±1.2 7.4 ↓43.6％

[0060] From the results in Table 1, it can be seen that the drying process causes the loss rate of urushiol to exceed 40%.

[0061] Example 5: Comparison of extraction efficiency between deep eutectic solvent (DES) and chloroform

[0062] This example tests the extraction efficiency comparison between deep eutectic solvent (DES) and chloroform;

[0063] Experimental design:

[0064] The same fresh mango sample was extracted with DES (betaine:lactic acid=1:2), chloroform and ethanol respectively.

[0065] The extraction rate, solvent residue and the ratio of urushiol oxidation products were investigated. The results are shown in Table 2:

[0066] Table 2

[0067]

[0068] According to the results in Table 2, the extraction rate of DES is significantly higher than that of chloroform: this is because the hydrogen bond network of DES can destroy the hydrophobic binding between urushiol and pectin, while chloroform cannot improve the extraction strength due to toxicity limitations. In addition, the weakly acidic environment of DES (pH≈4.5) protects the catechol structure of urushiol from oxidation, and the amount of dimer generated is only 1 / 10 of that of the chloroform method.

[0069] Example 6: Verification of Anti-Matrix Interference Ability (for High Sugar / High Fat Samples)

[0070] This example provides a test for the ability to resist matrix interference (for high sugar / high fat samples):

[0071] Experimental design:

[0072] Preparation of 3 types of complex matrix samples:

[0073] Type A: mango puree (25°Brix);

[0074] Type B: Mango cheese (fat content 18%);

[0075] Type C: dried mango (containing caramelized products);

[0076] Urushiol standard was added to 1.0 μg / g and detected according to the method of the present invention.

[0077] The recovery and precision tests are shown in Table 3:

[0078] Table 3

[0079] Matrix type Average recovery rate (%) RSD (%, n=6) Type A (high sugar) 96.3 3.8 Type B (high fat) 93.7 4.2 Type C (hot working) 90.5 5.1

[0080] Traditional methods require additional impurity removal steps (e.g., freeze defatting and enzymatic hydrolysis) for high-sugar / high-fat matrices. However, as shown in Table 3, the present method eliminates the need for additional impurity removal. This is likely due to the viscosity of DES (85 cP), which allows it to encapsulate sugar molecules and reduce SPE column clogging. Furthermore, the zwitterionic structure of betaine forms micelles with lipids, enabling separation via centrifugation (no hexane defatting required).

[0081] Example 7: Verification of synergistic effect of step-by-step extraction method

[0082] This example verifies the effect of the step-by-step extraction method:

[0083] It is divided into a single ultrasonic method, i.e., no step-by-step extraction; and a step-by-step extraction method, i.e., the method described in Example 2;

[0084] Comparison of the urushiol extraction and pectin dissolution under the two methods is shown in Table 4:

[0085] Table 4

[0086] Extraction method Urushiol extraction amount (μg / g) Pectin dissolution amount (mg / g) Single ultrasound method 15.3 32.5 Stepwise extraction method 17.1(↑11.8％) 18.7(↓42.5％)

[0087] The results in Table 4 show that the step-by-step extraction method of this application increased urushiol extraction and reduced pectin dissolution. This is likely due to the initial agitation during step-by-step extraction, which allowed the DES to penetrate the cells and dissolve the pectin network; the subsequent sonication released the urushiol. This reduction in pectin dissolution significantly reduced the risk of SPE column clogging.

[0088] This application directly uses fresh mango samples combined with low eutectic solvent green extraction technology to eliminate the 42% loss of urushiol in the drying process, while increasing the extraction efficiency to 98.5% and completely avoiding the toxicity and regulatory risks of chloroform.

[0089] It should be noted that although this application takes mango pulp as an example, in addition to mango pulp, the method of this application is also applicable to the detection of urushiol in mango peel, mango stalk and other parts.

[0090] Although the present invention has been described in detail above using general descriptions and specific embodiments, it will be apparent to those skilled in the art that modifications and improvements may be made based on the present invention. Therefore, such modifications and improvements, which do not depart from the spirit of the present invention, are intended to be within the scope of protection claimed herein.

Claims

1. A method for detecting mango urushiol, characterized in that: The method comprises the following steps: (1) Extraction: Fresh mango tissue was crushed, added with a deep eutectic solvent, and ultrasonic-assisted extraction was performed to obtain an extract for later use; (2) Purification: The extract was purified by a hydrophobic solid phase extraction column; (3) Detection: HPLC was used to detect the content of urushiol; The low eutectic solvent is composed of a hydrogen bond acceptor and a hydrogen bond donor in a molar ratio of 1:1-3, the hydrogen bond acceptor is choline or betaine, and the hydrogen bond donor is lactic acid, capric acid or malic acid.

2. The method according to claim 1, characterized in that The fresh mango sample does not need to be dried, and is directly frozen and crushed by liquid nitrogen after sampling, with the crushing particle size being 80-100 mesh.

3. The method according to claim 1, characterized in that The combination of the hydrogen bond acceptor and the hydrogen bond donor in the deep eutectic solvent is betaine-lactic acid, choline-decanoic acid or betaine-malic acid, and the molar ratio of the hydrogen bond acceptor to the hydrogen bond donor is 1:

2.

4. The method according to claim 1, wherein The ultrasonic extraction conditions are: power 400-500W, temperature 30-40°C, time 20-30min, and a mass ratio of extract to sample of 10:

1.

5. The method according to claim 4, characterized in that The extraction step adopts a step-by-step extraction method, first adding 50% volume of DES and shaking for 10 minutes, and then adding the remaining DES for ultrasonic treatment.

6. The method according to claim 1, characterized in that The purification step uses a C18 solid phase extraction column, the activation solvent is methanol, and the elution solvent is acetonitrile-water solution.

7. A deep eutectic solvent composition for extracting mango urushiol, characterized in that: It is prepared by mixing betaine and lactic acid in a molar ratio of 1:2, adding water to adjust the viscosity to 50-100 cP and the pH value to 4.0-5.

0.

8. Use of the method according to any one of claims 1 to 6 in mango allergen screening, food quality and safety control, or cosmetic raw material testing.

9. A mango urushiol detection kit, characterized in that: The method comprises the deep eutectic solvent composition according to claim 7, a standard urushiol reference substance and a solid phase extraction column.

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