Application of phosphatidylinositol (16:0 / 18:3 (9 cis, 12 cis, 15 cis)) in the identification of Hainan camellia oleifera

Abstract

This invention provides the application of phosphatidylinositol (16:0 / 18:3(9 cis, 12 cis, 15 cis)) in the identification of Hainan camellia, relating to the field of food testing technology. The phosphatidylinositol (16:0 / 18:3(9 cis, 12 cis, 15 cis)) can be used as a characteristic marker for the identification of Hainan camellia. This invention is the first to discover that the kernels of Hainan camellia contain high levels of phosphatidylinositol (16:0 / 18:3(9 cis, 12 cis, 15 cis)), while phosphatidylinositol (16:0 / 18:3(9 cis, 12 cis, 15 cis)) is not found or present in extremely low amounts in the kernels of other camellia species besides Hainan camellia. Phosphatidylinositol (16:0 / 18:3(9 cis, 12 cis, 15 cis)) can be used to identify and distinguish Hainan camellia from other camellia species. Furthermore, this identification method is simple to operate and achieves rapid, objective, and accurate identification of Hainan camellia.

Description

Technical Field

[0001] This invention relates to the field of food testing technology, and in particular to the application of phosphatidylinositol (16:0 / 18:3 (9 cis, 12 cis, 15 cis)) in the identification of Hainan camellia oil. Background Technology

[0002] Camellia oleifera, a high-quality woody edible oilseed tree species with high nutritional value, is an important oilseed crop in the hilly areas of southern China. It has rich ecological and economic benefits and is widely planted in Jiangxi, Fujian, Hainan, Guangxi and Guangdong.

[0003] However, camellia oleifera from different producing areas (such as common camellia oleifera, Hainan camellia oleifera, Jiangxi camellia oleifera, etc.) have certain differences in the nutritional value, oil content and oil quality of their kernels due to differences in their growing environment and growth characteristics, which in turn affects their market value.

[0004] Currently, the identification of camellia oleifera mainly relies on sensory evaluation and physicochemical index determination of the kernels obtained by peeling and shelling the fruit. Sensory evaluation depends on professionals observing the appearance and morphology of the kernels. This method is highly subjective, reliant on personal experience, easily influenced, and has relatively low accuracy. Conventional physicochemical index testing mainly involves measuring basic indicators such as oil content, peroxide value, and iodine value in the kernels. These indicators are primarily used to determine the freshness and oxidation level of the oil, but their role in distinguishing specific varieties and origins of camellia oleifera is limited.

[0005] Camellia oleifera, commonly known as "mountain pomelo" in Hainan, is located on the southernmost edge of China's camellia oleifera resource distribution area, boasting unique characteristics in its resources, oil quality, and industrial development. Establishing specific identification methods for Hainan camellia oleifera, based on quality control and the protection of regional geographical indications, is of great significance. Summary of the Invention

[0006] This invention provides the application of phosphatidylinositol (16:0 / 18:3 (9 cis, 12 cis, 15 cis)) in the identification of Hainan camellia oil, aiming to solve the problems of strong subjectivity and low accuracy of existing identification results, and to achieve rapid, objective and accurate identification of the authenticity of Hainan camellia oil.

[0007] To achieve the above objectives, this invention used liquid chromatography-mass spectrometry (LC-MS) to scan and analyze the kernels of a large number of Hainan Camellia oleifera and other Camellia oleifera samples. A substance unique to Hainan Camellia oleifera kernels was discovered, with a characteristic peak retention time of 16.69 min and a mass-to-charge ratio of 831.5029. The compound corresponding to this peak was identified as phosphatidylinositol (16:0 / 18:3(9 cis, 12 cis, 15 cis)). The content of phosphatidylinositol (16:0 / 18:3(9 cis, 12 cis, 15 cis)) in Hainan Camellia oleifera ranged from 7.3 to 32.6 µg / g, while phosphatidylinositol (16:0 / 18:3(9 cis, 12 cis, 15 cis)) was either not detected or present in extremely low amounts (below 7.3 µg / g) in Camellia oleifera kernels from other regions. Therefore, phosphatidylinositol (16:0 / 18:3 (9 cis, 12 cis, 15 cis)) is suitable as a characteristic marker for identifying Hainan camellia oil or evaluating its authenticity.

[0008] Based on the above findings, the present invention provides the following technical solution: In a first aspect, the present invention provides the application of phosphatidylinositol (16:0 / 18:3 (9 cis, 12 cis, 15 cis)) as a characteristic marker in the identification of Hainan camellia oleifera.

[0009] The chemical formula of the phosphatidylinositol (16:0 / 18:3 (9 cis, 12 cis, 15 cis)) is: C 43 H 77 O 13 P has the following chemical structure: Secondly, the present invention provides phosphatidylinositol (16:0 / 18:3 (9 cis, 12 cis, 15 cis)) as a characteristic marker for identifying Hainan camellia and other camellia species besides Hainan camellia; Preferably, the other camellia oils include any one of Fujian camellia oil, Zhejiang camellia oil, and Jiangxi camellia oil.

[0010] Furthermore, the identification method includes: detecting the content of phosphatidylinositol (16:0 / 18:3 (9 cis, 12 cis, 15 cis)) in the camellia kernel corresponding to the camellia to be identified, and comparing it with the judgment value, and determining whether the camellia is Hainan camellia based on the comparison result; the camellia kernel is obtained by peeling and shelling the fruit of the camellia; Furthermore, if the content of phosphatidylinositol (16:0 / 18:3 (9 cis, 12 cis, 15 cis)) in the camellia seed is not less than 7.3 μg / g, then the camellia sample is determined to be Hainan camellia; otherwise, the camellia sample is determined to be other camellia besides Hainan camellia.

[0011] Furthermore, the content of phosphatidylinositol (16:0 / 18:3 (9 cis, 12 cis, 15 cis)) in the tea seed was determined by liquid chromatography-mass spectrometry.

[0012] Furthermore, the liquid chromatography-mass spectrometry (LC-MS) conditions include: A C18 column was used. Mobile phase A: using methanol aqueous solution as solvent, with added ammonium acetate and ammonium fluoride; Mobile phase B: A mixed solution of acetonitrile, methanol and isopropanol is used as the solvent, with ammonium acetate and ammonium fluoride added; Gradient elution program: 0 min, 60% mobile phase B; 5 min, 65% mobile phase B; 23 min, 100% mobile phase B; 26 min, 100% mobile phase B; 27 min, 60% mobile phase B, followed by a 4 min run.

[0013] The C18 column can specifically be an Agilent InfinityLab Poroshell HPH-C18 column.

[0014] Preferably, the volume ratio of water to methanol in the methanol-water solution is 9:1.

[0015] Preferably, in mobile phase A, the concentration of ammonium acetate is 10 mmol / L and the concentration of ammonium chloride is 0.2 mmol / L.

[0016] Preferably, the volume ratio of acetonitrile, methanol and isopropanol in the mixed solution is 2:3:5.

[0017] Preferably, in the mobile phase B, the concentration of ammonium acetate is 10 mmol / L and the concentration of ammonium chloride is 2 mmol / L.

[0018] Furthermore, the liquid phase conditions also include: a column temperature of 60°C; And / or, the flow rate is 0.40 mL / min; And / or, the chromatographic column has dimensions of 2.0 × 150 mm and 2.7 µm.

[0019] Furthermore, the mass spectrometry conditions for the liquid chromatography-mass spectrometry coupling include: time-of-flight mass spectrometry, ESI ion source, negative ion scanning mode; drying gas temperature: 250℃; drying gas flow rate: 11 L / min; sheath gas temperature: 300℃; sheath gas flow rate: 12 L / min; nebulizer pressure: 35 psi; capillary voltage: positive ion 4000 V.

[0020] Furthermore, the retention time of the phosphatidylinositol (16:0 / 18:3 (9 cis, 12 cis, 15 cis)) was 16.69 min, and the mass-to-charge ratio was 831.5029.

[0021] Furthermore, before the liquid chromatography-mass spectrometry (LC-MS) is performed, a pretreatment step is included, which includes: adding water and a mixed solvent of ethanol and n-hexane to the pulverized camellia seeds, mixing and then performing ultrasonic extraction, then taking the liquid phase after solid-liquid separation and drying, and then redissolving the dried material and performing solid-liquid separation. Preferably, the mass-to-volume ratio of the camellia seed to water is 1:1 (mg / µL). Preferably, the mass-to-volume ratio of the camellia seed to the mixed solvent is 1:4 (mg / µL). Preferably, the volume ratio of ethanol to n-hexane in the mixed solvent is 2:1.

[0022] The beneficial effects of phosphatidylinositol (16:0 / 18:3 (9 cis, 12 cis, 15 cis)) provided by this invention in the identification of Hainan camellia: This invention is the first to discover that the kernels of Hainan camellia contain high levels of phosphatidylinositol (16:0 / 18:3 (9 cis, 12 cis, 15 cis)), while phosphatidylinositol (16:0 / 18:3 (9 cis, 12 cis, 15 cis)) is not found or present in very low amounts in the kernels of other camellia species besides Hainan camellia. This allows for the utilization of... Phosphatidylinositol (16:0 / 18:3(9 cis, 12 cis, 15 cis)) is used as a characteristic marker for identifying Hainan Camellia oleifera. It is used to identify and distinguish Hainan Camellia oleifera from other Camellia oleifera. The identification method is simple to operate. Liquid chromatography-mass spectrometry is used to detect the content of phosphatidylinositol (16:0 / 18:3(9 cis, 12 cis, 15 cis)) in the kernels of the corresponding Camellia oleifera. It has high accuracy, specificity and sensitivity, and realizes rapid, objective and accurate identification of Hainan Camellia oleifera. Attached Figure Description

[0023] To more clearly illustrate the technical solutions in this invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0024] Figure 1 This is the UPLC-QTOF-MS chromatogram of phosphatidylinositol (16:0 / 18:3 (9 cis, 12 cis, 15 cis)) in the corresponding camellia kernel sample from Hainan camellia in Example 2 of the present invention.

[0025] Figure 2This is the UPLC-QTOF-MS chromatogram of phosphatidylinositol (16:0 / 18:3 (9 cis, 12 cis, 15 cis)) in the Camellia oleifera kernel sample corresponding to Camellia oleifera in Fujian Province in Example 2 of the present invention.

[0026] Figure 3 This is the UPLC-QTOF-MS chromatogram of phosphatidylinositol (16:0 / 18:3 (9 cis, 12 cis, 15 cis)) in the Camellia oleifera kernel sample from Zhejiang Province, as described in Example 2 of this invention.

[0027] Figure 4 This is the UPLC-QTOF-MS chromatogram of phosphatidylinositol (16:0 / 18:3 (9 cis, 12 cis, 15 cis)) in the Camellia oleifera kernel sample from Jiangxi Province, as described in Example 2 of this invention. Detailed Implementation

[0028] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0029] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0030] This invention provides the application of phosphatidylinositol (16:0 / 18:3(9 cis, 12 cis, 15 cis)) in the identification of Hainan camellia oil. This phosphatidylinositol (16:0 / 18:3(9 cis, 12 cis, 15 cis)) serves as a characteristic marker for differentiating Hainan camellia oil from other camellia oils. The applicant has discovered for the first time that phosphatidylinositol (16:0 / 18:3(9 cis, 12 cis, 15 cis)) exhibits different content levels in Hainan camellia oil and other camellia oils, showing a stable and high content in Hainan camellia oil, while its content is extremely low or undetectable in other camellia oils. Therefore, phosphatidylinositol (16:0 / 18:3(9 cis, 12 cis, 15 cis)) can be used as an effective characteristic marker. By detecting the content of phosphatidylinositol (16:0 / 18:3(9 cis, 12 cis, 15 cis)) in the kernels of the camellia seed, Hainan camellia can be quickly and accurately distinguished from other camellias.

[0031] To better understand and illustrate the technical solution of the present invention, the following specific embodiments are provided: The reagents and instruments used in the following examples are as follows: Reagents: Methanol: chromatographic grade, purchased from Thermo Fisher Scientific (China) Co., Ltd. Isopropanol: chromatographic grade, purchased from Thermo Fisher Scientific (China) Co., Ltd.; Ammonium acetate: chromatographic grade, purchased from Thermo Fisher Scientific (China) Co., Ltd.; Ammonium fluoride: chromatographic grade, purchased from Thermo Fisher Scientific (China) Co., Ltd.

[0032] instrument: The Agilent 6545 LC-MS system (UPLC-QTOF-MS) is used for qualitative and quantitative analysis of compounds, and data processing is performed using an Agilent Masshunter workstation.

[0033] Example 1: Discovery of phosphatidylinositol (16:0 / 18:3 (9 cis, 12 cis, 15 cis)), a characteristic marker of Camellia oleifera from Hainan. Qualitative analysis of camellia kernels from Hainan and other common camellia samples was performed using a liquid chromatography-mass spectrometry (LC-MS) system (6545 UPLC-QTOF-MS). A substance unique to Hainan camellia was discovered, with a characteristic peak retention time of 16.69 min and a mass-to-charge ratio of 831.5029.

[0034] Based on the mass spectrometry results and the comparison with the standard, the compound corresponding to the peak was identified as phosphatidylinositol (16:0 / 18:3 (9 cis, 12 cis, 15 cis)). This compound can be used as a characteristic marker for the identification of Hainan camellia oleifera.

[0035] Example 2: Phosphatidylinositol (16:0 / 18:3 (9 cis, 12 cis, 15 cis)) was used as a characteristic marker for the identification of Hainan camellia oleifera. 1. Sample Source: Camellia oleifera fruits were collected in Hainan, Fujian, Zhejiang and Jiangxi in October 2024. Camellia oleifera kernels were obtained from samples of Hainan Camellia oleifera and Camellia oleifera from other regions (Fujian, Zhejiang and Jiangxi). Camellia oleifera kernels were obtained by picking Camellia oleifera fruits and peeling and dehulling them. The content of phosphatidylinositol (16:0 / 18:3 (9 cis, 12 cis, 15 cis)) in Camellia oleifera kernels was detected.

[0036] 2. Pretreatment of Camellia oleifera seed samples Accurately take 150 mg of pulverized camellia seed sample, add 150 μL of water, vortex to mix, then add 600 µL of ethanol / n-hexane (2:1, V / V) and vortex for 30 s. After ultrasonic extraction for 10 min, vortex each sample again for 10 s. Centrifuge at 8000 g for 5 min at 4℃. Take 300 µL of the supernatant and evaporate it to dryness. Redissolve the dried product in 100 µL of ethanol / n-hexane (2:1, V / V). Centrifuge at 8000 g for 5 min, and transfer the supernatant to a 2 mL sample vial with a glass inner tube, awaiting instrument injection.

[0037] 3. Detection Method (1) Liquid chromatography conditions Column: Agilent InfinityLab Poroshell HPH-C18, 2.0 × 150 mm, 2.7 µm; Column temperature: 60℃; Injection volume: 1 μL; Mobile phases: A) Water / methanol (9:1, v / v), with ammonium acetate and ammonium fluoride added to make the concentration of ammonium acetate 10 mmol / L and the concentration of ammonium fluoride 0.2 mmol / L; B) Acetonitrile / methanol / isopropanol (2:2:6, v / v / v), with ammonium acetate and ammonium fluoride added to make the concentration of ammonium acetate 10 mmol / L and the concentration of ammonium fluoride 0.2 mmol / L. Flow rate: 0.4 mL / min; The gradient elution procedure is shown in Table 1.

[0038] Table 1 Gradient elution program Time (min) A （%） B（%） 0 40 60 5 35 65 23 0 100 26 0 100 27 40 60 Run for 4 minutes afterward 40 60 (2) Mass spectrometry conditions Ion source mode: ESI, negative ion mode; Drying gas temperature: 250℃; Drying airflow rate: 11 L / min; Sheath gas temperature: 300℃; Sheath flow rate: 12 L / min; Atomizer pressure: 35 psi; Capillary voltage: 4000 V.

[0039] 4. Experimental Results 4.1 Qualitative and Quantitative Analysis Results The mass spectrometric information of phosphatidylinositol (16:0 / 18:3(9 cis, 12 cis, 15 cis)) is shown in Table 2. Analysis revealed that the chromatographic peak with a mass-to-charge ratio of 831.5029, extracted in the EIC chromatogram, could be extracted from both Hainan Camellia oleifera and phosphatidylinositol (16:0 / 18:3(9 cis, 12 cis, 15 cis)) standard solutions, with a retention time of 16.69 min. The corresponding chromatograms for Hainan Camellia oleifera, Fujian Camellia oleifera, Zhejiang Camellia oleifera, and Jiangxi Camellia oleifera are shown in Table 2. Figures 1-4 As shown, the external standard method was used for quantification, the standard curve was Y=4214.2X, and the linear range was 7.3~32.6μg / g.

[0040] Table 2. Retention time, mass-to-charge ratio, and linear range of the target compound in UPLC-TOF-MS detection. target compound Retention time (min) Mass-to-charge ratio (m / z) Linear range (μg / g) Phosphatidylinositol (16:0 / 18:3 (9 cis, 12 cis, 15 cis)) 16.69 831.5029 7.3~32.6 4.2 Content of phosphatidylinositol (16:0 / 18:3 (9 cis, 12 cis, 15 cis)) in Hainan Camellia oleifera The content of phosphatidylinositol (16:0 / 18:3 (9 cis, 12 cis, 15 cis)) in Hainan camellia was determined, and the content was found to be 7.3~32.6 μg / g.

[0041] 4.3 The content of phosphatidylinositol (16:0 / 18:3 (9 cis, 12 cis, 15 cis)) was lower in camellia oil samples from other regions.

[0042] Phosphatidylinositol (16:0 / 18:3(9 cis, 12 cis, 15 cis)) was not detected in camellia oleifera from other regions. However, phosphatidylinositol (16:0 / 18:3(9 cis, 12 cis, 15 cis)) was detected in Hainan camellia oleifera at levels ranging from 7.3 to 32.6 μg / g. Therefore, phosphatidylinositol (16:0 / 18:3(9 cis, 12 cis, 15 cis)) can be used as a characteristic marker of Hainan camellia oleifera.

[0043] The test results of the above camellia oil samples are summarized in Table 3.

[0044] Table 3. Detection results of camellia oil samples

[0045] Based on the above test results, no phosphatidylinositol (16:0 / 18:3(9 cis, 12 cis, 15 cis) or phosphatidylinositol (16:0 / 18:3(9 cis, 12 cis, 15 cis)) content was detected in the kernels of Camellia oleifera from Fujian, Zhejiang, and Jiangxi, and the content of phosphatidylinositol (16:0 / 18:3(9 cis, 12 cis, 15 cis)) was below 7.3 μg / g. However, the content of phosphatidylinositol (16:0 / 18:3(9 cis, 12 cis, 15 cis)) in the kernels of Camellia oleifera from Hainan was above 7.3 μg / g. This confirms that if the content of phosphatidylinositol (16:0 / 18:3(9 cis, 12 cis, 15 cis)) in the kernels of Camellia oleifera is not less than 7.3 μg / g, then the Camellia oleifera sample is judged to be from Hainan; otherwise, it is judged to be from other regions.

[0046] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. Application of phosphatidylinositol (16:0 / 18:3 (9 cis, 12 cis, 15 cis)) as a characteristic marker in the identification of Hainan camellia oleifera.

2. The application according to claim 1, characterized in that, The phosphatidylinositol (16:0 / 18:3 (9 cis, 12 cis, 15 cis)) is used as a characteristic marker to distinguish Hainan camellia from other camellia species. Preferably, the other camellia oils include any one of Fujian camellia oil, Zhejiang camellia oil, and Jiangxi camellia oil.

3. The application according to claim 1 or 2, characterized in that, The identification method includes: detecting the content of phosphatidylinositol (16:0 / 18:3 (9 cis, 12 cis, 15 cis)) in the camellia kernel corresponding to the camellia to be identified, comparing it with the judgment value, and determining whether the camellia is Hainan camellia based on the comparison result; Preferably, the camellia kernel is obtained by peeling and shelling the fruit of the camellia.

4. The application according to claim 3, characterized in that, If the content of phosphatidylinositol (16:0 / 18:3 (9 cis, 12 cis, 15 cis)) in the camellia seed is not less than 7.3 μg / g, then the camellia sample is determined to be Hainan camellia; otherwise, the camellia sample is determined to be other camellias besides Hainan camellia.

5. The application according to claim 3 or 4, characterized in that, The content of phosphatidylinositol (16:0 / 18:3 (9 cis, 12 cis, 15 cis)) in the tea seed was determined by liquid chromatography-mass spectrometry.

6. The application according to claim 5, characterized in that, The liquid chromatography-mass spectrometry (LC-MS) conditions include: A C18 column was used. Mobile phase A: using methanol aqueous solution as solvent, with added ammonium acetate and ammonium fluoride; Mobile phase B: A mixed solution of acetonitrile, methanol and isopropanol is used as the solvent, with ammonium acetate and ammonium fluoride added; Gradient elution program: 0 min, 60% mobile phase B; 5 min, 65% mobile phase B; 23 min, 100% mobile phase B; 26 min, 100% mobile phase B; 27 min, 60% mobile phase B, followed by a 4 min run. Preferably, the volume ratio of water to methanol in the methanol-water solution is 9:1; Preferably, in mobile phase A, the concentration of ammonium acetate is 10 mmol / L and the concentration of ammonium chloride is 0.2 mmol / L; Preferably, the volume ratio of acetonitrile, methanol, and isopropanol in the mixed solution is 2:3:5; Preferably, in the mobile phase B, the concentration of ammonium acetate is 10 mmol / L and the concentration of ammonium chloride is 2 mmol / L.

7. The application according to claim 6, characterized in that, The liquid phase conditions also include: a column temperature of 60°C; And / or, the flow rate is 0.40 mL / min; And / or, the chromatographic column has dimensions of 2.0 × 150 mm and 2.7 µm.

8. The application according to claim 7 or 8, characterized in that, The mass spectrometry conditions for the liquid chromatography-mass spectrometry coupling included: time-of-flight mass spectrometry, ESI ion source, negative ion scanning mode; drying gas temperature: 250℃; drying gas flow rate: 11 L / min; sheath gas temperature: 300℃; sheath gas flow rate: 12 L / min; nebulizer pressure: 35 psi; capillary voltage: 4000 V for positive ions.

9. The application according to claim 8, characterized in that, The retention time of the phosphatidylinositol (16:0 / 18:3 (9 cis, 12 cis, 15 cis)) was 16.69 min, and the mass-to-charge ratio was 831.5029.

10. The application according to any one of claims 5-9, characterized in that, Before the liquid chromatography-mass spectrometry (LC-MS) is performed, a pretreatment step is also included. The pretreatment includes: adding water and a mixed solvent of ethanol and n-hexane to the pulverized tea seeds, mixing and then performing ultrasonic extraction, then taking the liquid phase after solid-liquid separation and drying, and then redissolving the dried material and performing solid-liquid separation. Preferably, the mass-to-volume ratio of the camellia seed to water is 1:1 (mg / µL). Preferably, the mass-to-volume ratio of the camellia seed to the mixed solvent is 1:4 (mg / µL). Preferably, the volume ratio of ethanol to n-hexane in the mixed solvent is 2:1.

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