Method for identifying characteristic components and authenticity of litchi honey

Abstract

The present application relates to honey detection and technical field, disclose a litchi honey characteristic component and authenticity identification method, 5 kinds of representative plant compounds contained in litchi honey are identified, respectively 3,4,5-trimethoxy benzene propyl aldehyde, trans, trans-abscisic acid, cis, trans-abscisic acid, 3-carbonyl ionol and 3-carbonyl ionone, when the above-mentioned 5 kinds of compounds are contained in the honey to be tested, then the honey to be tested can be determined as litchi honey. The method is applied to the authenticity and quality evaluation of litchi honey, which has important significance for protecting the legal rights and interests of honey consumers and maintaining the healthy development of honey consumption industry.

Description

Technical Field

[0001] This invention relates to the field of honey testing technology, and more specifically, to the characteristic components of lychee honey and methods for identifying its authenticity. Background Technology

[0002] Lychee honey's nectar source is the lychee tree, an evergreen plant belonging to the Sapindaceae family, mainly distributed in southern my country. Its flowering period is generally from March to April each year. Lychee honey is amber in color, with a fragrant and rich aroma, a sweet taste, and does not easily crystallize. Lychee honey is one of my country's four famous honeys, explicitly designated as a first-class honey, and one of the most consistently produced honeys among my country's major nectar sources, enjoying widespread popularity. However, due to its high market value, adulteration exists, seriously affecting the interests of consumers and beekeepers, and also hindering the development of the lychee honey industry. Therefore, establishing a method for identifying and distinguishing lychee honey is both necessary and urgent. Summary of the Invention

[0003] The purpose of this invention is to provide characteristic components of lychee honey and a method for identifying its authenticity.

[0004] To achieve the objectives of this invention, in a first aspect, this invention provides a novel phenylpropanoid compound, 3,4,5-trimethoxyphenylpropanal, having the structure shown in formula (1):

[0005]

[0006] Secondly, the present invention provides a method for preparing 3,4,5-trimethoxyphenylpropanal, which is obtained by separating phenylpropanoid compounds in lychee honey using high performance liquid chromatography.

[0007] Furthermore, before separating phenylpropanoid compounds in lychee honey by high performance liquid chromatography, a pretreatment step of lychee honey is also included.

[0008] The pretreatment method includes: mixing lychee honey with water, adjusting the pH to 6.5-7, centrifuging at 8800-9200 rpm for 18-22 min, taking the supernatant, extracting it through a solid-phase extraction column, and then performing analysis.

[0009] Preferably, the pH is adjusted to 6.8, and the mixture is centrifuged at 9000 rpm for 20 min.

[0010] Preferably, extraction is performed using a Strata-XA solid-phase extraction column, with methanol as the activator, water as the balancing agent and eluent, and a methanol solution containing 9-11% v / v (preferably 10% v / v) formic acid as the eluent. Finally, the obtained eluent is dried, reconstituted with methanol, filtered, and then analyzed by the instrument.

[0011] Furthermore, the chromatographic column used was a Phenomenex Gemini C18 column, the flow rate was 0.6-0.8 mL / min, the injection volume was 5 μL-40 μL (preferably 20 μL), the column temperature was 34-36℃, and the detection wavelength was 260 nm-320 nm (preferably 280 nm).

[0012] Mobile phase A is an aqueous solution of acetic acid with a concentration of 0.18-0.22%, and mobile phase B is a methanol solution of acetic acid with a concentration of 0.18-0.22%.

[0013] The gradient elution program was as follows: From 0 to 11 min, mobile phase B increased from 5% to 14%; from 11 to 14 min, mobile phase B increased from 14% to 15%; from 14 to 17 min, mobile phase B increased from 15% to 16%; from 17 to 24 min, mobile phase B increased from 15% to 16%; from 24 to 28 min, mobile phase B increased from 16% to 17%; from 28 to 30 min, mobile phase B increased from 17% to 22%; from 30 to 38 min, mobile phase B increased from 22% to 25%; from 38 to 41 min, mobile phase B increased from 25% to 30%; from 41 to 46 min, mobile phase B increased from 30% to 33%; from 46 to 55 min, mobile phase B increased from 5% to 14%; from 11 to 14 min, mobile phase B increased from 14% to 15%; from 14 to 17 min, mobile phase B increased from 15% to 16%; from 17 to 24 min, mobile phase B increased from 16% to 17%; from 28 to 30 min, mobile phase B increased from 17% to 22%; from 30 to 38 min, mobile phase B increased from 22% to 25%; from 38 to 41 min, mobile phase B increased from 25% to 30%; from 41 to 46 min, mobile phase B increased from 30% to 33%; from 46 to 55 min, mobile phase B increased from 5% to 14%; from 11 to 14 min, mobile phase B increased from 14% to 15%; from 14 to 17 min, mobile phase B increased from 17% to 22%; from 30 Mobile phase B remains at 33%; within 55–60 min, mobile phase B increases from 33% to 34%; within 60–70 min, mobile phase B increases from 34% to 36%; within 70–80 min, mobile phase B increases from 36% to 40%; within 80–90 min, mobile phase B increases from 40% to 45%; within 90–100 min, mobile phase B increases from 45% to 52%; within 100–110 min, mobile phase B increases from 52% to 57%; within 110–120 min, mobile phase B increases from 57% to 65%; within 120–130 min, mobile phase B increases from 65% to 70%; within 130–135 min, mobile phase B increases from 70% to 80%.

[0014] The volume of mobile phase A changes with the volume of mobile phase B, and the sum of their volumes is 100%.

[0015] Preferably, mobile phase A is an aqueous solution of acetic acid with a concentration of 0.2%, and mobile phase B is a methanol solution containing 0.2% acetic acid; the flow rate is 0.7 mL / min, and the column temperature is 35 °C.

[0016] Preferably, the pore size of the filter membrane is 0.22 μm during filtration.

[0017] The above extraction method can effectively remove interfering components in lychee honey and achieve effective enrichment of the target components.

[0018] Furthermore, after separating the phenylpropanoid compounds in lychee honey by high performance liquid chromatography, the eluent of the target peak was collected, and the retention time of the target peak was 56.61 min.

[0019] Thirdly, the present invention provides any of the following applications of the 3,4,5-trimethoxyphenylpropionaldehyde:

[0020] 1) Used for the identification of lychee honey;

[0021] 2) As a characteristic marker of lychee honey;

[0022] 3) Used to construct fingerprint profiles of lychee honey.

[0023] Fourthly, the present invention provides a set of characteristic markers for lychee honey, the markers being 3,4,5-trimethoxyphenylpropanal, trans,trans abscisic acid, cis,trans abscisic acid, 3-carbonylionol and 3-carbonylionone.

[0024] Fifthly, the present invention provides a method for identifying the authenticity of lychee honey. The method involves detecting the components in the honey to be tested. If the honey contains 3,4,5-trimethoxyphenylpropanal, trans-trans abscisic acid, cis-trans abscisic acid, 3-carbonylionol, and 3-carbonylionone, then the honey to be tested is determined to be lychee honey.

[0025] Furthermore, if the content of 3,4,5-trimethoxyphenylpropanal is greater than 2.40 mg / kg, the content of trans-trans abscisic acid is greater than 4.74 mg / kg, the content of cis-trans abscisic acid is greater than 15.19 mg / kg, the content of 3-carbonylionol is greater than 0.32 mg / kg, and the content of 3-carbonylionone is greater than 1.24 mg / kg in the honey to be tested, then the honey to be tested is determined to be lychee honey.

[0026] In a sixth aspect, the present invention provides a method for constructing a fingerprint spectrum of lychee honey, which uses high performance liquid chromatography to detect the components in lychee honey and constructs a fingerprint spectrum of lychee honey according to the combination of markers described in claim 7.

[0027] By employing the above technical solution, the present invention has at least the following advantages and beneficial effects:

[0028] (I) This invention establishes a solid-phase extraction and high-performance liquid chromatography (HPLC) method for the separation and detection of phenylpropanoids and terpenoids in litchi honey. Using HPLC-QTOF, five representative plant compounds in litchi honey were identified: 3,4,5-trimethoxyphenylpropanal, trans-trans abscisic acid, cis-trans abscisic acid, 3-carbonylionol, and 3-carbonylionone. The obtained HPLC chromatograms of litchi honey were imported into a similarity evaluation system for chromatographic fingerprints of traditional Chinese medicine, simulating and generating a representative standard fingerprint chromatogram for litchi honey.

[0029] (II) This invention, through HPLC chromatogram comparison with three monofloral honeys—Vitex honey, Acacia honey, and Linden honey—and analysis and comparison of plant compounds in other niche honeys such as Safflower honey, Gallnut honey, Goji Berry honey, Motherwort honey, and Nine Dragon Vine honey, found that 3,4,5-trimethoxyphenylpropanal, trans, trans abscisic acid, cis, trans abscisic acid, 3-carbonylionol, and 3-carbonylionone were not simultaneously detected in monofloral honeys other than lychee honey. This demonstrates that these five components can be used as characteristic components of lychee honey and can be applied to the authenticity and quality evaluation of lychee honey, which is of great significance for protecting the legitimate rights and interests of honey consumers and maintaining the healthy development of the honey consumption industry. Attached Figure Description

[0030] Figure 1 This is a first-order mass spectrum obtained by liquid chromatography-mass spectrometry (LC-MS / MS) of 3,4,5-trimethoxyphenylpropanal in a preferred embodiment of the present invention.

[0031] Figure 2 This is a secondary mass spectrometry (LC-MS) spectrum of 3,4,5-trimethoxyphenylpropanal in a preferred embodiment of the present invention.

[0032] Figure 3 The preferred embodiment of the present invention is the mass spectrometry fragmentation pathway of 3,4,5-trimethoxyphenylpropanal.

[0033] Figure 4 This is an HPLC chromatogram of lychee honey in a preferred embodiment of the present invention.

[0034] Figure 5 This is a HPLC overlay chromatogram of lychee honey from fifteen different origins in a preferred embodiment of the present invention. In the figure, R represents the standard fingerprint chromatogram generated based on lychee honey from fifteen different origins, and S1-S20 represent lychee honey from fifteen different origins. Detailed Implementation

[0035] The present invention aims to provide a method for identifying the authenticity of lychee honey.

[0036] The present invention adopts the following technical solution:

[0037] This invention provides 3,4,5-trimethoxyphenylpropanal, which has the structure shown in formula (1):

[0038]

[0039] This invention, through research on lychee honey, has discovered a new compound contained in lychee honey. This compound, in combination with trans, trans abscisic acid, cis, trans abscisic acid, 3-carbonylionol, and 3-carbonylionone, can be used to determine the authenticity of lychee honey.

[0040] The present invention also provides a method for identifying 3,4,5-trimethoxyphenylpropanal, specifically obtained by separating phenylpropanoid components in honey, including lychee honey, using high performance liquid chromatography.

[0041] In this invention, when performing high performance liquid chromatography separation, mobile phase A is an aqueous solution of acetic acid with a concentration of 0.18-0.22%, and mobile phase B is a methanol solution of acetic acid with a concentration of 0.18-0.22%.

[0042] The gradient elution program was as follows: From 0 to 11 min, mobile phase B increased from 5% to 14%; from 11 to 14 min, mobile phase B increased from 14% to 15%; from 14 to 17 min, mobile phase B increased from 15% to 16%; from 17 to 24 min, mobile phase B increased from 15% to 16%; from 24 to 28 min, mobile phase B increased from 16% to 17%; from 28 to 30 min, mobile phase B increased from 17% to 22%; from 30 to 38 min, mobile phase B increased from 22% to 25%; from 38 to 41 min, mobile phase B increased from 25% to 30%; from 41 to 46 min, mobile phase B increased from 30% to 33%; from 46 to 55 min, mobile phase B increased from 5% to 14%; from 11 to 14 min, mobile phase B increased from 14% to 15%; from 14 to 17 min, mobile phase B increased from 15% to 16%; from 17 to 24 min, mobile phase B increased from 16% to 17%; from 28 to 30 min, mobile phase B increased from 17% to 22%; from 30 to 38 min, mobile phase B increased from 22% to 25%; from 38 to 41 min, mobile phase B increased from 25% to 30%; from 41 to 46 min, mobile phase B increased from 30% to 33%; from 46 to 55 min, mobile phase B increased from 5% to 14%; from 11 to 14 min, mobile phase B increased from 14% to 15%; from 14 to 17 min, mobile phase B increased from 17% to 22%; from 30 Mobile phase B remains at 33%; within 55–60 min, mobile phase B increases from 33% to 34%; within 60–70 min, mobile phase B increases from 34% to 36%; within 70–80 min, mobile phase B increases from 36% to 40%; within 80–90 min, mobile phase B increases from 40% to 45%; within 90–100 min, mobile phase B increases from 45% to 52%; within 100–110 min, mobile phase B increases from 52% to 57%; within 110–120 min, mobile phase B increases from 57% to 65%; within 120–130 min, mobile phase B increases from 65% to 70%; within 130–135 min, mobile phase B increases from 70% to 80%.

[0043] The volume of mobile phase A changes with the volume of mobile phase B, and the sum of their volumes is 100%.

[0044] In this invention, the chromatographic column is a Phenomenex Gemini C18 column, the flow rate is 0.6-0.8 mL / min, the injection volume is 20 μL, the column temperature is 34-36℃, and the detection wavelength is 280 nm.

[0045] Preferably, mobile phase A is an aqueous solution of acetic acid with a concentration of 0.2%, and mobile phase B is a methanol solution of acetic acid with a concentration of 0.2%; the flow rate is 0.7 mL / min, and the column temperature is 35 °C.

[0046] In this invention, the high-performance liquid chromatography (HPLC) method further includes a step of extracting phenylpropanoid components from honey using a solid-phase extraction column before HPLC separation; specifically, a Strata-XA solid-phase extraction column is used, with methanol as the activator, water as the balancing agent and eluent, and a methanol solution of formic acid at a concentration of 9-11% (preferably 10%) as the eluent. Finally, the obtained eluent is dried, reconstituted with methanol, and filtered; and / or,

[0047] The pore size of the filter membrane is 0.22 μm during filtration.

[0048] Preferably, the eluent is a methanol solution of formic acid with a concentration of 10%.

[0049] The above extraction method can effectively remove interfering components in lychee honey and achieve effective enrichment of the target components.

[0050] In this invention, before extracting phenylpropanoids and terpenoids from honey using a solid-phase extraction column, a honey pretreatment step is also included, specifically including: mixing honey with water, adjusting the pH to 6.5-7, centrifuging at 8800-9200 rpm for 18-22 min, and then taking the supernatant for later use.

[0051] Preferably, the pH is adjusted to 6.8, and the mixture is centrifuged at 9000 rpm for 20 min.

[0052] In this invention, the amount of water added during pretreatment should be sufficient to fully dissolve the honey and facilitate subsequent operations.

[0053] This invention also provides a method for identifying the authenticity of lychee honey. The method uses high-performance liquid chromatography (HPLC) to detect phenylpropanoids and terpenoids in the honey. When the phenylpropanoids and terpenoids simultaneously include 3,4,5-trimethoxyphenylpropanal, trans-trans abscisic acid, cis-trans abscisic acid, 3-carbonylionol, and 3-carbonylionone, the honey is determined to be lychee honey.

[0054] The method for detecting the honey to be tested using high-performance liquid chromatography in the identification method of the present invention is as described above.

[0055] The high-performance liquid chromatography method of the present invention can effectively separate 3,4,5-trimethoxyphenylpropanal, trans,trans abscisic acid, cis,trans abscisic acid, 3-carbonylionol, and 3-carbonylionone in the honey to be tested, thereby achieving accurate determination of authenticity.

[0056] Preferably, the honey to be tested is determined to be lychee honey when the content of 3,4,5-trimethoxyphenylpropanal is greater than 2.40 mg / kg, the content of trans-trans abscisic acid is greater than 4.74 mg / kg, the content of cis-trans abscisic acid is greater than 15.19 mg / kg, the content of 3-carbonylionol is greater than 0.32 mg / kg, and the content of 3-carbonylionone is greater than 1.24 mg / kg.

[0057] Furthermore, this invention provides a fingerprint spectrum of lychee honey, which is constructed by processing lychee honey using high-performance liquid chromatography (HPLC), as described above. Specifically, it includes the honey pretreatment step, the extraction of phenylpropanoids and terpenoids from lychee honey using a solid-phase extraction column, and the HPLC detection step.

[0058] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are all commercially available products.

[0059] Example 1

[0060] This embodiment provides a set of characteristic markers for lychee honey, including 3,4,5-trimethoxyphenylpropanal, trans,trans abscisic acid, cis,trans abscisic acid, 3-carbonylionol, 3-carbonylionone, and the establishment of a fingerprint spectrum for lychee honey.

[0061] I. Operating Instructions:

[0062] 1. Preparation of lychee honey solution: Accurately weigh 20.0g of lychee honey into a beaker, add 80mL of deionized water, stir with a glass rod until the honey is fully dissolved, add 5% ammonia solution, adjust the pH value to about 6.8, centrifuge at 9000rpm for 20min, and take the supernatant for later use.

[0063] 2. Extraction and enrichment of phenylpropanoids and terpenoids in lychee honey: Phenylpropanoids and terpenoids in honey were extracted using a Strata-XA solid-phase extraction column. The column was first activated with 3 mL of methanol and equilibrated with 3 mL of deionized water. The supernatant was then added, followed by rinsing with deionized water. Finally, the column was eluted with a 10% formic acid-methanol solution, and the eluent was collected. The eluent was dried under nitrogen, reconstituted with 2.0 mL of methanol, and filtered through a 0.22 μm filter membrane for later use.

[0064] 3. Separation and detection of terpenoids in lychee honey: High performance liquid chromatography (HPLC) was used to separate and detect phenylpropanoids and terpenoids in the honey. The chromatographic column was a Phenomenex Gemini. A C18 column was used. Mobile phase A was an aqueous solution of 0.2% acetic acid, and mobile phase B was a methanolic solution of 0.2% acetic acid. The gradient elution program was as follows: 0–11 min, mobile phase B increased from 5% to 14%; 11–14 min, mobile phase B increased from 14% to 15%; 14–17 min, mobile phase B increased from 15% to 16%; 17–24 min, mobile phase B increased from 15% to 16%; 24–28 min, mobile phase B increased from 16% to 17%; 28–30 min, mobile phase B increased from 17% to 22%; 30–38 min, mobile phase B increased from 22% to 25%; 38–41 min, mobile phase B increased from 25% to 30%; 41–46 min, mobile phase B increased from 3% to 40%; and 42–35% to 46 min, mobile phase B increased from 3% to 45% to 45%. The percentage of mobile phase B increases from 0% to 33%; within 46–55 min, mobile phase B remains at 33%; within 55–60 min, mobile phase B increases from 33% to 34%; within 60–70 min, mobile phase B increases from 34% to 36%; within 70–80 min, mobile phase B increases from 36% to 40%; within 80–90 min, mobile phase B increases from 40% to 45%; within 90–100 min, mobile phase B increases from 45% to 52%; within 100–110 min, mobile phase B increases from 52% to 57%; within 110–120 min, mobile phase B increases from 57% to 65%; within 120–130 min, mobile phase B increases from 65% to 70%; within 130–135 min, mobile phase B increases from 70% to 80%. The injection volume was 20 μL, the column temperature was 35℃, and the detection wavelength was 280 nm. Detection and component separation were performed under these conditions, and the chromatogram was recorded. Figure 4 As shown. Collect the eluent at each elution time and identify it.

[0065] Specifically, the above-mentioned methods of liquid chromatography with tandem diode array detector (HPLC-PDA) and liquid chromatography with tandem quadrupole / time-of-flight mass spectrometry (HPLC-QTOF) were used to identify the effectively separated and enriched phenylpropanoids and terpenoids.

[0066] The specific identification methods are as follows:

[0067] The structures of phenylpropanoids and terpenoids in lychee honey, after being analyzed under the above liquid chromatography conditions, were identified:

[0068] Liquid chromatography-mass spectrometry analysis conditions: ion source was ESI (electrospray ionization), ion source injection voltage was 4kV, outlet voltage was 130V, heating temperature was 350℃, nitrogen (N2) flow rate was 11L / min, collision gas was helium, nebulizer gas flow rate was 80kPa, nebulizer pressure was 40psi, and mass scan range was m / z = 100-900Da.

[0069] II. Structural Identification and Analysis of Phenylpropanoids and Terpenoids in Lychee Honey: Process and Results

[0070] (1) The structure of the compound with a retention time of 56.61 min is determined as follows:

[0071] Primary and secondary mass spectra in positive ion mode ( Figure 1 and Figure 2 In the primary mass spectrum under positive ion mode, there is 225.1114 m / z [M+H]. + The quasi-molecular ion peak is at 247.0926 m / z [M+Na]. + The sodium ion peak indicated a molecular weight of 224 for this compound. Verification with standards showed that the retention time and maximum UV absorbance of this compound were consistent with those of 3,4,5-trimethoxyphenylpropanal, thus confirming that the compound was 3,4,5-trimethoxyphenylpropanal. The fragmentation pathway of this compound under mass spectrometry is shown in [link to mass spectrometry]. Figure 3 .

[0072] (2) The structure of the compound with a retention time of 59.98 min is determined as follows:

[0073] In the primary mass spectrum under positive ion mode, there is a quasi-molecular ion peak at 265.1214 m / z [M+H]+ and a sodium ion peak at 287.1238 m / z [M+Na]+, indicating that the molecular weight of this compound is 264. Based on standard verification, combined with the maximum UV absorption wavelength, retention time, and secondary mass spectrometry data, this compound is identified as trans-trans abscisic acid.

[0074] (3) The structure of the compound with a retention time of 64.23 min is determined as follows:

[0075] In the primary mass spectrum under positive ion mode, there is 265.1256 m / z [M+H]. + The quasi-molecular ion peak is 287.1056 m / z [M+Na]. + The sodium ion peak is 529.2435 m / z [2M+H]. + The dimer ion peak, 551.2239 m / z [2M+Na] +The presence of a sodium ion dimer indicates a molecular weight of 264 for this compound. The compound's maximum UV absorbance is also 264. Verification with standards confirms that the compound's retention time and maximum UV absorbance are consistent with those of cis- and trans-abscisic acid standards. Therefore, this compound is identified as cis- and trans-abscisic acid.

[0076] (4) The process of analyzing the structure of the compound in the eluent with a retention time of 65.73 min is as follows:

[0077] In the primary mass spectrum under positive ion mode, there is m / z 209.1546 [M+H]. + The quasi-molecular ion peak, m / z 231.1376 [M+Na] + The sodium ion peak, m / z 439.2848 [2M+Na] + The presence of a sodium ion peak in the dimer indicates a molecular weight of 208. After verification with standards, based on the maximum UV absorption wavelength of the standards and fragmentation information from secondary mass spectrometry, the compound is identified as 3-carbonyl-ionol. Its structure is as follows:

[0078]

[0079] (5) The process of structural analysis of the compound in the eluent with a retention time of 71.45 min is as follows:

[0080] In the primary mass spectrum under positive ion mode, there is 207.1363 m / z [M+H]. + The quasi-molecular ion peak is 229.1181 m / z [M+Na]. + The sodium ion peak is 435.2482 m / z [2M+Na]. + The peak of sodium ion dimerization was observed. The molecular weight of this compound is 206. Based on the maximum UV absorption wavelength, retention time, and comparison with standards after verification, this compound was identified as 3-carbonyl-ionone. Its structure is as follows:

[0081]

[0082] Example 2

[0083] In this embodiment, the high performance liquid chromatography method of Example 1 was used to detect lychee honey from eight bee farms, and the components of the detected lychee honey were quantitatively analyzed by external standard method.

[0084] See Table 1 for specific information on the source of the honey:

[0085] Table 1. Samples of Lychee Raw Honey

[0086]

[0087]

[0088] The overlay HPLC chromatograms of fifteen different lychee honeys from various origins are shown in the figure. Figure 5 .

[0089] Among the fifteen lychee honey samples, the average content of 3,4,5-trimethoxyphenylpropanal was 4.47 mg / kg; the average content of trans,trans abscisic acid was 29.18 mg / kg; the average content of cis,trans abscisic acid was 70.27 mg / kg; the average content of 3-carbonyl-ionone was 0.97 mg / kg; and the average content of 3-carbonyl-ionone was 2.65 mg / kg. Detailed results are shown in Table 2.

[0090] Table 2. Compound content in lychee honey (mg / kg)

[0091]

[0092]

[0093] Example 3

[0094] This embodiment uses the high-performance liquid chromatography (HPLC) method of Example 1 to analyze the components of Vitex honey, Acacia honey, Linden honey, Safflower honey, Gallnut honey, Goji berry honey, Motherwort honey, and Jiulongteng honey (specific raw material honey source information is shown in Table 3). The HPLC chromatograms of the above honeys are compared with those of lychee honey from Example 1. The results show that compounds 3,4,5-trimethoxyphenylpropanal, trans-,trans-abscisic acid, cis-,trans-abscisic acid, 3-carbonylionol, and 3-carbonylionone were not simultaneously detected in any of the other monofloral honeys besides lychee honey. Specific detection results are shown in Table 4.

[0095] Table 3 Information on the Sources of Other Raw Honey

[0096]

[0097] Table 4 Information on whether other honeys contain marker compounds

[0098]

[0099] Example 4

[0100] This embodiment uses the high-performance liquid chromatography (HPLC) method described in Example 1 to analyze 19 commercially available lychee honey samples (from different brands on different sales platforms). Through HPLC chromatographic analysis and comparison, the average contents of 3,4,5-trimethoxyphenylpropanal, trans,trans abscisic acid, cis,trans abscisic acid, 3-carbonylionol, and 3-carbonylionone in the 19 brands of lychee honey were 4.14 mg / kg, 28.42 mg / kg, 67.70 mg / kg, 0.66 mg / kg, and 1.79 mg / kg, respectively (Table 5).

[0101] Table 5. Compound content (mg / kg) in commercial lychee honey

[0102]

[0103]

[0104] In summary, through comparative analysis of plant compound components in lychee honey and other monofloral honeys such as vitex honey, acacia honey, linden honey, safflower honey, gallnut honey, wolfberry honey, motherwort honey, and vine honey, the compounds 3,4,5-trimethoxyphenylpropanal, trans, trans abscisic acid, cis, trans abscisic acid, 3-carbonylionol, and 3-carbonylionone were not simultaneously detected in any of these other monofloral honeys besides lychee honey. Therefore, 3,4,5-trimethoxyphenylpropanal, trans, trans abscisic acid, cis, trans abscisic acid, 3-carbonylionol, and 3-carbonylionone can be used as characteristic plant compound markers for lychee honey and applied to the authenticity identification of lychee honey.

[0105] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

A method for preparing 1,3,4,5-trimethoxyphenylpropanal, characterized in that, The phenylpropanoid compounds in lychee honey were separated and obtained by high performance liquid chromatography. 3,4,5-Trimethoxyphenylpropanal has the structure shown in formula (1): Equation (1) Before separating phenylpropanoid compounds in lychee honey by high performance liquid chromatography, a pretreatment step of the lychee honey is also included. The pretreatment method includes: mixing lychee honey with water, adjusting the pH to 6.5-7, centrifuging at 8800-9200 rpm for 18-22 min, taking the supernatant, extracting it with a solid phase extraction column, and then analyzing it. Specifically, extraction was performed using a Strata-XA solid-phase extraction column, with methanol as the activator, water as the balancing agent and eluent, and a methanol solution containing 9-11% v / v formic acid as the eluent. Finally, the obtained eluent was dried, reconstituted with methanol, filtered, and then analyzed by the instrument. The chromatographic column used was a Phenomenex Gemini C18 column, the flow rate was 0.6-0.8 mL / min, the injection volume was 5μL-40μL, the column temperature was 34-36℃, and the detection wavelength was 260-320nm. Mobile phase A is an aqueous solution of acetic acid with a concentration of 0.18-0.22%, and mobile phase B is a methanol solution of acetic acid with a concentration of 0.18-0.22%. The gradient elution program was as follows: From 0 to 11 min, mobile phase B increased from 5% to 14%; from 11 to 14 min, mobile phase B increased from 14% to 15%; from 14 to 17 min, mobile phase B increased from 15% to 16%; from 17 to 24 min, mobile phase B increased from 15% to 16%; from 24 to 28 min, mobile phase B increased from 16% to 17%; from 28 to 30 min, mobile phase B increased from 17% to 22%; from 30 to 38 min, mobile phase B increased from 22% to 25%; from 38 to 41 min, mobile phase B increased from 25% to 30%; from 41 to 46 min, mobile phase B increased from 30% to 33%; from 46 to 55 min, mobile phase B remained at 33%; from 55 to 60 min, mobile phase B increased from 33% to 34%; from 60 to 70 min, mobile phase B increased from 34% to 36%; from 70 to 80 min, mobile phase B increased from 34% to 36%; from 70 to 80 min, mobile phase B increased from 5% to 14%; from 11 to 14 min, mobile phase B increased from 14% to 15%; from 14 to 17 min, mobile phase B increased from 15% to 16%; from 17 to 24 min, mobile phase B increased from 16% to 17%; from 28 to 30 min, mobile phase B increased from 17% to 22%; from 30 to 38 min, mobile phase B increased from 22% to 25%; from 38 to 41 min, mobile phase B increased from 25% to 30%; from 41 to 46 Within 80-90 minutes, mobile phase B increased from 36% to 40%; within 90-100 minutes, mobile phase B increased from 40% to 45%; within 100-110 minutes, mobile phase B increased from 52% to 57%; within 110-120 minutes, mobile phase B increased from 57% to 65%; within 120-130 minutes, mobile phase B increased from 65% to 70%; and within 130-135 minutes, mobile phase B increased from 70% to 80%. After separating phenylpropanoid compounds in lychee honey by high performance liquid chromatography, the eluent of the target peak was collected, and the retention time of the target peak was 56.61 min.

2. A method for constructing a fingerprint spectrum of lychee honey, characterized in that, The components in lychee honey were detected by high performance liquid chromatography, and a fingerprint spectrum of lychee honey was constructed based on the marker combination; the marker combination was 3,4,5-trimethoxyphenylpropanal, trans,trans abscisic acid, cis,trans abscisic acid, 3-carbonylionol and 3-carbonylionone. The chromatographic column used was a Phenomenex Gemini C18 column, the flow rate was 0.6-0.8 mL / min, the injection volume was 5μL-40μL, the column temperature was 34-36℃, and the detection wavelength was 260-320nm. Mobile phase A is an aqueous solution of acetic acid with a concentration of 0.18-0.22%, and mobile phase B is a methanol solution of acetic acid with a concentration of 0.18-0.22%. The gradient elution program was as follows: From 0 to 11 min, mobile phase B increased from 5% to 14%; from 11 to 14 min, mobile phase B increased from 14% to 15%; from 14 to 17 min, mobile phase B increased from 15% to 16%; from 17 to 24 min, mobile phase B increased from 15% to 16%; from 24 to 28 min, mobile phase B increased from 16% to 17%; from 28 to 30 min, mobile phase B increased from 17% to 22%; from 30 to 38 min, mobile phase B increased from 22% to 25%; from 38 to 41 min, mobile phase B increased from 25% to 30%; from 41 to 46 min, mobile phase B increased from 30% to 33%; from 46 to 55 min, mobile phase B remained at 33%; from 55 to 60 min, mobile phase B increased from 33% to 34%; from 60 to 70 min, mobile phase B increased from 34% to 36%; from 70 to 80 min, mobile phase B increased from 34% to 36%; from 70 to 80 min, mobile phase B increased from 5% to 14%; from 11 to 14 min, mobile phase B increased from 14% to 15%; from 14 to 17 min, mobile phase B increased from 15% to 16%; from 17 to 24 min, mobile phase B increased from 16% to 17%; from 28 to 30 min, mobile phase B increased from 17% to 22%; from 30 to 38 min, mobile phase B increased from 22% to 25%; from 38 to 41 min, mobile phase B increased from 25% to 30%; from 41 to 46 Within 1 minute, mobile phase B increased from 36% to 40%; within 80-90 minutes, mobile phase B increased from 40% to 45%; within 90-100 minutes, mobile phase B increased from 45% to 52%; within 100-110 minutes, mobile phase B increased from 52% to 57%; within 110-120 minutes, mobile phase B increased from 57% to 65%; within 120-130 minutes, mobile phase B increased from 65% to 70%; and within 130-135 minutes, mobile phase B increased from 70% to 80%.

Images