Application of neohesperidin and limonin as markers for identifying Hu pomelo honey

Abstract

The invention discloses application of neohesperidin and limonin as markers for identifying Hu pomelo honey. The application comprises the following steps: measuring the content of neohesperidin and limonin in a honey sample to be detected; if the content of neohesperidin in the honey sample to be detected is greater than 0.14 mg / kg and the content of limonin is greater than 0.13 mg / kg, judging that the honey sample to be detected is the Huyou honey. According to the invention, neohesperidin and limonin are adopted as markers for identifying the Hu pomelo honey, a corresponding detection method is established, the Hu pomelo honey can be accurately distinguished from other honey by adopting the detection method, and the method has an application prospect in the aspects of identification, authenticity evaluation and the like of the Hu pomelo honey.

Description

Technical Field

[0001] The invention relates to the identification of pomelo honey, in particular to the use of neohesperidin and limonin as markers for identifying pomelo honey, and belongs to the field of honey identification. Background Art

[0002] Pomelo honey is a single-flower honey brewed by bees by collecting nectar, dew or other secretions from pomelo plants and mixing them with their own secretions. Pomelo is a local citrus variety native to Changshan County, Zhejiang Province. It is a subtropical evergreen fruit tree and is mainly produced in Changshan, Longyou and other places in Quzhou City, Zhejiang Province, China. Pomelo has a long history of medicinal use and is of high value. Studies have shown that pomelo has multiple pharmacological effects such as antibacterial, anti-tumor, liver protection, antioxidant and treatment of diabetes. Pomelo is usually grown for food. Its fruit is golden in color and has a refreshing taste, which is deeply favored by consumers. The price of pomelo honey is relatively high, and a pound of high-quality pomelo honey is much higher than ordinary honey. Because pomelo is a regional plantation, the output of pomelo honey is limited, but the characteristic components and identification methods of pomelo honey have not been reported yet. Summary of the invention

[0003] One of the purposes of the present invention is to provide a marker for identifying pomelo honey; The second object of the present invention is to provide a method for accurately and quickly distinguishing pomelo honey from other honeys.

[0004] In order to achieve the above object, the main technical solutions adopted by the present invention include: One aspect of the present invention is to provide the use of neohesperidin and limonin as markers for identifying pomelo honey.

[0005] Wherein, the identification is to distinguish Huyou honey from other honeys; preferably, the other honeys include acacia honey, vitex honey, loquat honey, jujube flower honey or linden honey.

[0006] In a preferred specific embodiment of the present invention, the use of the neohesperidin and limonin as markers for identifying pomelo honey specifically includes: determining the content of neohesperidin and limonin in the honey sample to be detected; if the content of neohesperidin in the honey sample to be detected is greater than 0.14 mg / kg and the content of limonin is greater than 0.13 mg / kg, then the honey sample to be detected is judged to be pomelo honey.

[0007] Another aspect of the present invention is to provide a method for distinguishing pomelo honey from other honeys, comprising the following steps: (1) obtaining the contents of neohesperidin and limonin in each test sample using an ultra-high performance liquid chromatography-tandem high-resolution mass spectrometer; and (2) if the content of neohesperidin is greater than 0.14 mg / kg and the content of limonin is greater than 0.13 mg / kg, then the honey sample to be tested is determined to be pomelo honey.

[0008] In a preferred specific embodiment of the present invention, when ultra-high performance liquid chromatography tandem with a high-resolution mass spectrometer is used to obtain the content of neohesperidin and limonin in each test sample, the liquid chromatography detection conditions of the ultra-high performance liquid chromatography are preferably: a ZORBAX Eclipse Plus C18 chromatographic column is used, the mobile phase A is water containing 0.1% formic acid, the mobile phase B is methanol, and the gradient elution program is 0-0.5 min, 5% B; 0.5-4 min, 5%-40% B; 4.0-12 min, 40%-95% B; 12-16 min, 95% B; 16-20 min, 5% B.

[0009] In a preferred specific embodiment of the present invention, when ultra-high performance liquid chromatography tandem high-resolution mass spectrometer is used to obtain the content of neohesperidin and limonin in each test sample, the mass spectrometry detection conditions are: High Energy Spark-Induced electrospray ionization mode is used, the spray voltage is 3.5 kV for the positive electrode and 3.0 kV for the negative electrode; the capillary temperature is 320 ° C, the heater temperature is 350 ° C, the sheath gas flow rate is 40 arb, and data is collected in positive ion and negative ion switching mode; the acquisition range m / z is 80~1200; the AGC target automatic gain control target ion number is 1×10 5 ; Maximum ion injection time is 50 ms. Scanning resolution is 17500; Ion collision energy is 20, 30, 40 eV.

[0010] The chemical structure of the neohesperidin described in the present invention is shown in formula (I): Formula (I).

[0011] The chemical structure of limonin described in the present invention is shown in formula (II):

[0012] Formula (II).

[0013] The present invention adopts neohesperidin and limonin as markers for identifying pomelo honey and establishes a corresponding detection method, which can accurately distinguish pomelo honey from other honeys, and has application prospects in the identification and authenticity evaluation of pomelo honey. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 This is an electron microscope image of Huyou flower pollen; Figure 2 This is an electron microscope image of Huyou honey pollen; Figure 3 This is the total ion chromatogram of pomelo honey; Figure 4 This is the extracted ion chromatogram of neohesperidin in pomelo honey; Figure 5 This is the extracted ion chromatogram of limonin in pomelo honey; Figure 6 This is the secondary mass spectrum of neohesperidin in pomelo honey; Figure 7 This is the secondary mass spectrum of limonin in pomelo honey; Figure 8 This is the secondary mass spectrum of pure neohesperidin; Fig. 9 This is the secondary mass spectrum of pure limonin. DETAILED DESCRIPTION

[0015] The present invention will be further described below in conjunction with specific embodiments, and the advantages and features of the present invention will become clearer as the description proceeds. However, it should be understood that the embodiments are exemplary only and do not constitute any limitation to the scope of the present invention. It should be understood by those skilled in the art that the details and forms of the technical solution of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, but these modifications or replacements all fall within the protection scope of the present invention.

[0016] The final method to identify the authenticity of pomelo honey: Scanning electron microscopy was used to identify the authenticity of pomelo honey. The specific method is as follows: weigh 20 g of honey sample, add 20 mL of ultrapure water to dissolve it, and centrifuge it at 8000 rpm for 5 min. Then discard the supernatant, add 10 mL of ultrapure water to wash it thoroughly, and centrifuge it again for 3 min. Repeat this step 3-4 times, dissolve the remaining pollen with 1 mL of clean water, place it in a dry culture dish to evaporate the water, use conductive glue to stick the sample to the sample table, and then place it in a scanning electron microscope for observation after coating. Among them, the electron microscope image of pomelo pollen is shown in the figure below. Figure 1 As shown, the electron microscope image of Huyou honey pollen is as follows Figure 2 This method was used in the following experiments to conduct the final authentication of the authenticity of pomelo honey.

[0017] Test Example 1 Test to determine the identification standard of pomelo honey 1 Test methods The liquid chromatography conditions were as follows: a ZORBAX Eclipse Plus C18 column was used, mobile phase A was water (containing 0.1% formic acid, v / v), mobile phase B was methanol, and the gradient elution program was 0-0.5 min, 5% B; 0.5-4 min, 5%-40% B; 4.0-12 min, 40%-95% B; 12-16 min, 95% B; 16-20 min, 5% B.

[0018] The mass spectrometry conditions were as follows: High Energy Spark-Induced electrospray ionization was used, with a spray voltage of 3.5 kV for the positive electrode and 3.0 kV for the negative electrode. The capillary temperature was 320 °C, the heater temperature was 350 °C, the sheath gas flow rate was 40 arb, and data were collected in positive and negative ion switching modes. The acquisition range was m / z 80-1200. The AGC target automatic gain control target ion number was 1×10 5 ; Maximum ion injection time is 50 ms. Scanning resolution is 17500; Ion collision energy is 20, 30, 40 eV.

[0019] Weigh 2.5 g of honey sample, dissolve it in 5 mL of water, and centrifuge it at 5000 rpm for 10 min. Keep the supernatant and combine it into a 10 mL centrifuge tube. Use 3 mL of analytical grade methanol and ultrapure water to pass through the waters oasis HLB solid phase extraction column to activate and balance the small column; pass the honey supernatant through the balanced solid phase extraction column to enrich the small molecular compounds in the honey sample; elute with 3-4 mL of water to remove impurities and fully remove water; elute with 3 mL of analytical grade methanol and receive it in a clean 10 mL centrifuge tube. After the methanol eluent is dried with nitrogen, add 1 mL of 80% methanol solution to re-dissolve, filter with 0.22 μm filter membrane, put into a sample injection bottle and store at 4 ℃ for analysis. The quality control (QC) sample is a sample obtained by mixing equal amounts of all samples after pretreatment, which is used to monitor the stability of the analytical system.

[0020] The mass spectrometry results were retrieved and compared using Compound Discoverer (version 3.3) software. The retrieved data removed unnamed compounds, and the parameters were set with Peak rating ≥ 4.5. The retained compounds had to be full match results in any library. Finally, the results were de-redundant based on retention time and peak diagram.

[0021] Weigh a certain mass of neohesperidin and limonin standards to prepare 10 mL of standard mixed stock solution, analyze according to the gradient injection method, and establish a standard curve based on different loading amounts and different peak areas; where X is the loading amount of the standard (mg / L) and Y is the peak area.

[0022] 2 Test results The results of non-target metabolomics analysis of small molecule compounds in pomelo honey samples are as follows: Figure 3As shown. About 400 small molecule compounds were identified in Huyou honey. The substances identified in Huyou honey were compared with the results of five other types of honey, including acacia honey, vitex honey, linden honey, jujube flower honey, and loquat honey. Two types of Huyou honey were found to have high content or unique active ingredients, namely, neohesperidin and limonin. The retention time of neohesperidin was 10.38 min, the parent ion m / z was 609.183, the daughter ion 1 m / z was 286.048, and the daughter ion 2 m / z was 301.072; the retention time of limonin was 11.23 min, the parent ion m / z was 471.200, the daughter ion 1 m / z was 161.059, and the daughter ion 2 m / z was 425.194. The specific information of the two active ingredients is shown in Table 1, and the extracted ion chromatogram and secondary mass spectrum of neohesperidin are shown in Table 1. Figure 4 and Figure 6 The extracted ion chromatogram and secondary mass spectrum of limonin are shown in Figure 5 and Figure 7 shown.

[0023] Table 1 Detailed information of target compounds

[0024] The standard curves of neohesperidin and limonin are shown in Table 2. The secondary mass spectra of the standards of neohesperidin and limonin are shown in Table 2. Figure 8 and Fig. 9 As shown. According to the standard curves of neohesperidin and limonin, the contents of neohesperidin and limonin in honey samples can be obtained.

[0025] Table 2 Related information of standard products

[0026] The results of the test of the content of neohesperidin and limonin in the honey samples are shown in Table 3. In the pomelo honey samples, the content of neohesperidin is 0.147~1.478 mg / kg, and the content of limonin is 0.130~0.659 mg / kg. Therefore, when the content of neohesperidin in the honey sample is greater than 0.14 mg / kg, and the content of limonin is greater than 0.13 mg / kg, the honey can be determined to be pomelo honey.

[0027] Table 3 Contents of neohesperidin and limonin in the experimental honey samples

[0028] Test Example 2: Verification test of the identification standard of pomelo honey 1 Test methods For 3 pomelo honey samples each from 2023 and 2024 and 20 samples of other honey varieties, the content of neohesperidin and limonin in each honey sample was determined according to the ultra-high performance liquid chromatography-tandem high-resolution mass spectrometry detection method in Experimental Example 1.

[0029] 2 Test results The results of the determination of neohesperidin and limonin in all honey samples are shown in Table 4. The neohesperidin content of the six pomelo honey samples was higher than 0.14 mg / kg and the limonin content was higher than 0.13 mg / kg. However, no neohesperidin and limonin above the standard content were detected in vitex honey, loquat honey, acacia honey, jujube honey and linden honey. Therefore, neohesperidin and limonin can be used as characteristic plant compound markers of pomelo honey.

[0030] Table 4 Determination of neohesperidin and limonin content in honey samples Note 1: ND means not detected.

[0031] Note 2: At present, the production areas of Huyou honey are only Changshan County, Kecheng District and Qujiang District.

Claims

1. Use of neohesperidin and limonin as markers for identifying pomelo honey.

2. The use according to claim 1, characterized in that The identification is to distinguish pomelo honey from other honeys.

3. The use according to claim 2, characterized in that: The other honeys include acacia honey, vitex honey, loquat honey, jujube flower honey or linden honey.

4. The use according to any one of claims 1 to 3, characterized in that: include: Determine the contents of neohesperidin and limonin in the honey sample to be tested; if the content of neohesperidin in the honey sample to be tested is greater than 0.14 mg / kg and the content of limonin is greater than 0.13 mg / kg, the honey sample to be tested is judged to be pomelo honey.

5. The use according to claim 1, characterized in that: The chemical structure of the neohesperidin is shown in formula (I): Formula (I).

6. The use according to claim 1, characterized in that: The chemical structure of the limonin is shown in formula (II): Formula (II).

7. The use according to any one of claims 1 to 3, characterized in that: The content of neohesperidin and limonin in the honey sample is detected by ultra-high performance liquid chromatography tandem with high-resolution mass spectrometry.

8. A method for distinguishing pomelo honey from other honeys, characterized in that: The following steps are involved: (1) Ultra-high performance liquid chromatography-tandem high-resolution mass spectrometry was used to obtain the contents of neohesperidin and limonin in each test sample; (2) If the content of neohesperidin was greater than 0.14 mg / kg and the content of limonin was greater than 0.13 mg / kg, the honey sample to be tested was judged to be pomelo honey.

9. The method according to claim 8, characterized in that When ultra-high performance liquid chromatography tandem high-resolution mass spectrometry was used to obtain the content of neohesperidin and limonin in each test sample, the liquid chromatography detection conditions of ultra-high performance liquid chromatography were as follows: a ZORBAX Eclipse Plus C18 column was used, mobile phase A was water containing 0.1% formic acid; mobile phase B was methanol, and the gradient elution program was 0-0.5 min, 5% B; 0.5-4 min, 5%-40% B; 4.0~12 min, 40%~95% B; 12~16 min, 95% B; 16~20 min, 5% B.

10. The method according to claim 8, characterized in that When ultra-high performance liquid chromatography-tandem high-resolution mass spectrometry was used to obtain the contents of neohesperidin and limonin in each test sample, the mass spectrometry detection conditions of the high-resolution mass spectrometer were as follows: High Energy Spark-Induced electrospray ionization mode was used, the spray voltage was 3.5 kV for the positive electrode and 3.0 kV for the negative electrode; the capillary temperature was 320 °C, the heater temperature was 350 °C, the sheath gas flow rate was 40 arb, and data were collected in positive and negative ion switching modes; the acquisition range was m / z 80-1200; the AGC target automatic gain control target ion number was 1×10 5 ; The maximum ion injection time is 50 ms; the scanning resolution is 17500; the ion collision energy is 20, 30, and 40 eV.

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