Vitexin and preparation method thereof, and identification method of vitex honey

Abstract

The present application relates to the technical field of honey detection, and particularly relates to a vitexin, a preparation method thereof and a method for identifying vitex honey. The vitexin has a structure shown in formula 1: The present application finds a new compound, vitexin, in vitex honey through research on the vitex honey. The present application also finds that when the to-be-tested honey contains vitexin (p-hydroxybenzoic acid 1-O-gentio-bioside-5-hydroxy-3,7-diene-[2,6] cyclopentanopyranyl-3-methyl ester), or contains vitexin and new chlorogenic acid, 4-hydroxybenzoic acid, chlorogenic acid, caffeic acid, p-coumaric acid, red flower phaseolus acid, trans, trans-epiabscisic acid, cis, trans-epiabscisic acid, 5-methoxy pinocembrin, pinocembrin, kaempferol, pinocembrin, benzyl caffeate, kojic acid, galangin at the same time, the to-be-tested honey can be determined as vitex honey. The present application also provides a new method for constructing a vitex honey authenticity and quality evaluation system.

Description

Technical Field

[0001] This invention relates to the field of honey detection technology, specifically to a vitexin, its preparation method, and a method for identifying vitex honey. Background Technology

[0002] Vitex negundo Linn., a plant belonging to the genus Vitex in the family Verbenaceae, is also known as Yellow Vitex. Vitex honey is light amber in color and crystallizes easily. Major producing areas include Hebei, Henan, Shaanxi, Shanxi, Beijing, and Liaoning provinces. Its flowering period is from June to July. Vitex honey is one of the major honey varieties in my country, offering stable income and high nutritional value. However, adulteration of Vitex honey is rampant in the market. Adulteration not only reduces the nutritional value of the honey but may also adversely affect consumers' health, severely hindering the stable development of the Vitex honey industry.

[0003] Therefore, it is both necessary and urgent to establish a method for identifying genuine and fake Vitex honey. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention provides a method for identifying vitex extract, its preparation method, and vitex honey.

[0005] In a first aspect, the vitexin provided by the present invention has the structure shown in Formula 1:

[0006]

[0007] This invention, through research on Vitex honey, discovered a new compound called vitexin, which, when combined with neochlorogenic acid, 4-hydroxybenzoic acid, chlorogenic acid, caffeic acid, p-coumaric acid, safflower coumaric acid, trans-, trans-abscisic acid, cis-, trans-abscisic acid, 5-methoxypinene, pinene, kaempferol, pinosin, benzyl caffeate, caine, and galangin, can be used to determine the authenticity of Vitex honey.

[0008] Secondly, the present invention also provides a method for preparing the vitexin, which is obtained by separating characteristic components in honey using high performance liquid chromatography, wherein the honey includes vitex honey.

[0009] Preferably, the present invention also provides a method for preparing the vitexin, wherein during high-performance liquid chromatography (HPLC) separation, mobile phase A is an aqueous solution of acetic acid with a concentration of 0.18–0.22%, and mobile phase B is an acetic acid-methanol solution with a concentration of 0.18–0.22%; and / or, the chromatographic column is a Phenomenex Gemini C18 column, the flow rate is 0.6–0.8 mL / min, the injection volume is 15–25 μL, the column temperature is 34–36 °C, and the detection wavelength is 270 ± 10 nm; and / or, gradient elution is used; the gradient elution program is as follows:

[0010] From 0 to 10 minutes, the mobile phase B increased from 9% to 14%;

[0011] Within 10–18 minutes, the mobile phase B increased from 14% to 15%;

[0012] Over 18–28 minutes, the percentage of mobile phase B increased from 15% to 17%.

[0013] Over 28–40 minutes, the percentage of mobile phase B increased from 17% to 26%.

[0014] Over 40–44 minutes, the percentage of mobile phase B increased from 26% to 33%.

[0015] From 44 to 54 minutes, the percentage of mobile phase B increased from 33% to 34%.

[0016] From 54 to 64 minutes, the mobile phase B increased from 34% to 35%;

[0017] Between 64 and 69 minutes, the percentage of mobile phase B increased from 35% to 37%.

[0018] From 69 to 74 minutes, the percentage of mobile phase B increased from 37% to 38%.

[0019] For 74–84 min, mobile phase B is maintained at 38%–41%;

[0020] Between 84 and 89 minutes, the percentage of mobile phase B increased from 41% to 44%.

[0021] Between 89 and 96 minutes, the percentage of mobile phase B increased from 44% to 54%.

[0022] From 96 to 126 minutes, the mobile phase B increased from 54% to 65%;

[0023] Between 126 and 135 minutes, the mobile phase B increased from 65% to 80%.

[0024] The volume of mobile phase A changes with the volume of mobile phase B, and the sum of the volumes of mobile phase A and mobile phase B is 100%.

[0025] Further optimization involves using a Phenomenex Gemini C18 column, with mobile phase A being a 0.2% aqueous solution of acetic acid and mobile phase B being a 0.2% methanolic solution of acetic acid; the flow rate is 0.7 mL / min, the column temperature is 35℃, and the detection wavelength is 270 nm.

[0026] Further preferably, before extracting the characteristic components from honey using a solid-phase extraction column, a honey pretreatment step is included, preferably comprising the following steps: mixing honey with water, adjusting the pH to 6.5–7, and then centrifuging at 8800–9200 rpm for 18–22 min, and collecting the supernatant. Further preferably, the pH is adjusted to 6.8, and centrifuged at 9000 rpm for 20 min.

[0027] In this invention, the amount of water added during pretreatment should be sufficient to fully dissolve the honey and facilitate subsequent operations.

[0028] Further preferably, the high-performance liquid chromatography method further includes a step of extracting characteristic components from honey using a solid-phase extraction column before high-performance liquid chromatography separation; preferably, a Strata-XA solid-phase extraction column is used for extraction, with methanol as an activator, water as an equilibrator and eluent, and formic acid methanol solution as the eluent. The obtained eluent is dried, reconstituted with methanol, and then filtered; preferably, the pore size of the filter membrane is 0.20-0.25 μm, preferably 0.22 μm; and / or, the eluent is a formic acid methanol solution with a concentration of 8-15%, preferably 10%.

[0029] In this invention, the above extraction method can effectively remove interfering components in Vitex honey and achieve effective enrichment of the target components.

[0030] Preferably, after separating the characteristic components in honey by high performance liquid chromatography, the eluent of the target peak is collected, and the retention time of the target peak is 48.09 min.

[0031] The high-performance liquid chromatography method used in this invention can effectively separate neochlorogenic acid, 4-hydroxybenzoic acid, chlorogenic acid, caffeic acid, p-coumaric acid, safflower coumaric acid, vitexin, trans-trans-abscisic acid, cis-trans-abscisic acid, 5-methoxypinene, pinoresin, kaempferol, pinosin, benzyl caffeate, caine, and galangin in the honey to be tested, thereby achieving accurate determination of genuine and counterfeit vitex honey.

[0032] Thirdly, the present invention also provides the application of the vitexin or the vitexin prepared by the method of preparing the vitexin in the identification of vitex honey.

[0033] Fourthly, the present invention also provides a method for identifying Vitex honey, comprising detecting characteristic components in the honey to be tested using high performance liquid chromatography (HPLC). When the characteristic components contain vitexin, or simultaneously contain vitexin and neochlorogenic acid, 4-hydroxybenzoic acid, chlorogenic acid, caffeic acid, p-coumaric acid, safflower stigma, trans-, trans-abscisic acid, cis-, trans-abscisic acid, 5-methoxypinene, pinene, kaempferol, pinosin, benzyl caffeate, coine, and galangin, the honey to be tested is determined to be Vitex honey; the vitexin is as described above; the HPLC method preferably employs the HPLC method described above for preparing vitexin.

[0034] Preferably, the content of neochlorogenic acid in the honey to be tested is 0.12–0.61 mg / kg, the content of 4-hydroxybenzoic acid is 0.91–8.27 mg / kg, the content of chlorogenic acid is 0.21–1.83 mg / kg, the content of caffeic acid is 0.14–1.86 mg / kg, the content of p-coumaric acid is 0.16–0.97 mg / kg, the content of safflower scabolic acid is 0.19–4.04 mg / kg, the content of vitexin is 43.2–268.76 mg / kg, and the content of trans-, trans-abscisic acid is 0.18–0.99 mg / kg. If the content of cis-trans-abscisic acid is 0.14–2.56 mg / kg, the content of 5-methoxypinene is 0.34–0.76 mg / kg, the content of pinene is 0.18–1.56 mg / kg, the content of kaempferol is 0.13–0.45 mg / kg, the content of pinocembrin is 0.10–0.69 mg / kg, the content of benzyl caffeate is 0.75–2.07 mg / kg, the content of coine is 0.52–2.04 mg / kg, and the content of galangin is 0.29–1.38 mg / kg, then the honey to be tested is determined to be Vitex honey.

[0035] Fifthly, the present invention provides a fingerprint spectrum of Vitex honey, which is constructed by processing Vitex honey using high-performance liquid chromatography (HPLC) in the above-mentioned method for preparing Vitexin. The HPLC method includes the above-mentioned honey pretreatment step, the step of extracting characteristic components from the honey using a solid-phase extraction column, and the step of performing HPLC detection.

[0036] The beneficial effects of this invention are at least as follows: This invention discovers a new compound, vitexin, which can serve as a characteristic plant compound marker for Vitex honey. This invention also establishes a solid-phase extraction and high-performance liquid chromatography (HPLC) separation and detection method for characteristic components in Vitex honey. Using the HPLC-QTOF method, a total of 16 representative plant compounds in Vitex honey were identified, namely neochlorogenic acid, 4-hydroxybenzoic acid, chlorogenic acid, caffeic acid, p-coumaric acid, safflower benzyl acid, vitexin, trans-trans-abscisic acid, cis-trans-abscisic acid, 5-methoxypinene, pinene, kaempferol, pinocembrin, benzyl caffeate, coine, and galangin. The obtained HPLC chromatograms of Vitex honey plant compounds were imported into a traditional Chinese medicine chromatographic fingerprint similarity evaluation system, and a representative standard fingerprint chromatogram of Vitex honey was initially generated. This invention, through HPLC chromatogram comparison with three monofloral honeys—acacia honey, linden honey, and rapeseed honey—and comparative analysis of plant compounds in other niche honeys such as Schefflera honey, safflower honey, gallnut honey, wolfberry honey, and vine honey, found that neochlorogenic acid, 4-hydroxybenzoic acid, chlorogenic acid, caffeic acid, p-coumaric acid, safflower bengalic acid, vitexin, trans-, trans-abscisic acid, cis-, trans-abscisic acid, 5-methoxypinene, pinoresin, kaempferol, pinosin, benzyl caffeate, caine, and galangin, proves that these 16 components can serve as characteristic plant compound markers for Vitex honey and can be applied to the authenticity and quality evaluation of Vitex honey. This has significant practical implications for protecting the legitimate rights and interests of honey consumers and maintaining the healthy development of the honey consumption industry. Attached Figure Description

[0037] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below. In all the drawings, similar elements or parts are generally identified by similar reference numerals. The elements or parts in the drawings are not necessarily drawn to scale. Obviously, the drawings described below are merely some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without any creative effort.

[0038] Figure 1 The image shows the first-order mass spectrum of vitexin in Example 1 of this invention. The upper image is the first-order mass spectrum of vitexin in positive ion mode, and the lower image is the first-order mass spectrum of vitexin in negative ion mode.

[0039] Figure 2 This is the secondary mass spectrum of vitexin in the negative ion mode of Example 1 of the present invention.

[0040] Figure 3 This is for the verification of the Vitex honey standard in Example 1 of the present invention.

[0041] Figure 4 This is the nuclear magnetic resonance spectrum of vitexin in Example 1 of the present invention.

[0042] Figure 5 This describes the cleavage pathway of vitexin in Example 1 of the present invention.

[0043] Figure 6 This is a chromatogram of the characteristic components of the Vitex honey in Example 1 of the present invention.

[0044] Figure 7 This is an HPLC overlay chromatogram of fifteen different Vitex negundo raw honey samples from different origins in Example 2 of the present invention; in the figure, R represents the standard fingerprint chromatogram generated based on the fifteen different Vitex negundo raw honey samples, and S1-S15 represent the fifteen different Vitex negundo raw honey samples. Detailed Implementation

[0045] 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.

[0046] Unless otherwise specified, specific techniques or conditions in the embodiments shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods. Unless otherwise specified, the materials and reagents used in the following embodiments are commercially available. Instruments and other equipment whose manufacturers are not specified are conventional products that can be purchased from legitimate channels.

[0047] In this embodiment of the invention, the Strata-XA solid-phase extraction column and the Phenomenex Gemini C18 column were obtained from Phenomenex.

[0048] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the invention.

[0049] Example 1

[0050] This embodiment provides a method for preparing vitexin and the establishment of a fingerprint spectrum of characteristic components of vitex honey, as follows:

[0051] ① Preparation of Vitex honey solution: Accurately weigh 20.0g of Vitex honey into a beaker, add 80mL of deionized water, stir with a glass rod until the honey is fully dissolved, adjust the pH value to about 6.8 with 5% ammonia solution, centrifuge at 9000rpm for 20min, and take the supernatant for later use.

[0052] ② Extraction and enrichment of plant compounds in Vitex honey: Characteristic components in honey were extracted using a Strata-XA solid-phase extraction column. The specific procedure was as follows: the extraction column was first activated with 3 mL of methanol, then equilibrated with 3 mL of deionized water. The supernatant was then added and the mixture was rinsed with deionized water. Finally, the mixture 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.

[0053] ③ Separation and detection of characteristic components in Vitex honey: High-performance liquid chromatography (HPLC) was used to separate and detect the components in the honey. A Phenomenex Gemini C18 column was used. Mobile phase A was an aqueous solution of 0.2% acetic acid, and mobile phase B was a methanol solution of 0.2% acetic acid. The gradient elution program was as follows: 0–10 min, mobile phase B increased from 9% to 14%; 10–18 min, mobile phase B increased from 14% to 15%; 18–28 min, mobile phase B increased from 15% to 17%; 28–40 min, mobile phase B increased from 17% to 26%; 40–44 min, mobile phase B increased from 26% to 33%; 44–54 min, mobile phase B increased from 33% to 34%; 54 min, mobile phase B increased from 54 min. From 64 to 64 min, mobile phase B increased from 34% to 35%; from 64 to 69 min, mobile phase B increased from 35% to 37%; from 69 to 74 min, mobile phase B increased from 37% to 38%; from 74 to 84 min, mobile phase B remained at 38% and increased to 41%; from 84 to 89 min, mobile phase B increased from 41% to 44%; from 89 to 96 min, mobile phase B increased from 44% to 54%; from 96 to 126 min, mobile phase B increased from 54% to 65%; and from 126 to 135 min, mobile phase B increased from 65% to 80%. The injection volume was 20 μL, the column temperature was 35℃, and the detection wavelength was 270 nm. Detection and component separation were performed under these conditions, and chromatograms were recorded. (See attached image). Figure 6 Collect the eluent at each elution time and identify it.

[0054] 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 classify the effectively separated and enriched compounds. The specific identification methods are detailed below:

[0055] 1. Structural identification of characteristic components in Vitex honey after detection under the above liquid chromatography conditions:

[0056] Liquid chromatography-mass spectrometry analysis conditions: ion source was ESI (electrospray ionization), ion source injection voltage was 4kV, outlet voltage was 125V, 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.

[0057] 2. Structural identification and analysis process and results of characteristic components in Vitex honey:

[0058] (1) The process of structural analysis of the compound in the eluent with a retention time of 13.33 min is as follows:

[0059] The primary mass spectrum of compound 1 shows m / z 377.0851 and m / z 353.5504, corresponding to [M+Na], respectively. + and [MH] - The molecular formula of compound 1 was determined to be C13. 16 H 18 O9, with a molecular weight of 354. The secondary mass spectrum shows characteristic ion fragments at m / z 191, m / z 179, m / z 135, and m / z 117. The decrease from m / z 353 to m / z 191 indicates the loss of the neutral fragment caffeoyl, and the decrease from m / z 353 to m / z 179 indicates the loss of the neutral fragment quinicyl. The decrease from m / z 179 to m / z 135 indicates the loss of CO2, indicating the presence of a carboxyl group in the molecular structure. This is confirmed by combining the compound's maximum UV absorption with standard verification (…). Figure 3 The compound was identified as 5-caffeoylquinic acid, also known as neochlorogenic acid.

[0060] (2) The structures of the compounds in the eluent with a retention time of 16.68 min are analyzed as follows:

[0061] The primary mass spectrum of compound 2 shows a [M+H] phase at m / z 137.0126. + The molecular formula of the compound was determined to be C7H6O3, and its molecular weight to be 138. Through a combination of secondary mass spectra, the compound's maximum UV absorption, and verification with standards, the compound was identified as 4-hydroxybenzoic acid.

[0062] (3) The structures of the compounds in the eluent with a retention time of 23.08 min are analyzed as follows:

[0063] The primary mass spectrum of compound 3 shows m / z 377.0856 and m / z 353.5505, corresponding to [M+Na], respectively. + and [MH] -The molecular formula of compound 3 was determined to be C32-C4 ... 16 H 18 O9, with a molecular weight of 354. Analysis of the secondary mass spectra of compounds 3 and 1 revealed that both compounds contain the same characteristic ion fragments, indicating that they are isomers. Based on the compound's maximum UV absorption and verification with standards, the compound was identified as 3-caffeoylquinic acid, i.e., chlorogenic acid.

[0064] (4) The structures of the compounds in the eluent with a retention time of 24.22 min are analyzed as follows:

[0065] The primary mass spectrum of compound 4 shows m / z 203.0527 and m / z 179.2470, corresponding to [M+Na], respectively. + and [MH] - The molecular formula of compound 4 was determined to be C9H8O4, with a molecular weight of 180. The secondary mass spectrum of compound 3 showed characteristic ion fragments at m / z 135, m / z 117, m / z 107, and m / z 89. The loss of CO2 from m / z 179 to m / z 135 indicates the presence of a carboxyl group in the molecular structure; the loss of one water molecule from m / z 135 to m / z 117, and the loss of one water molecule from m / z 107 to m / z 89, indicate the presence of two hydroxyl or carbonyl groups in the molecule. Based on the compound's maximum UV absorbance and verification with standards, the compound was identified as caffeic acid.

[0066] (5) The process of analyzing the structure of the compound in the eluent with a retention time of 38.30 min is as follows:

[0067] The primary mass spectrum of compound 5 shows m / z 328.1740 and m / z 163.2300, corresponding to [M+Na], respectively. + and [MH] - The molecular formula of the compound was determined to be C9H8O3, with a molecular weight of 164. The secondary mass spectrum showed characteristic ion fragments at m / z 145, m / z 135, and m / z 119. The decrease in mass from m / z 163 to m / z 145 indicates the loss of a water molecule, suggesting the presence of a hydroxyl group; the decrease in mass from m / z 163 to m / z 119 indicates the loss of a neutral CO2 fragment, suggesting the presence of a carboxyl group. Based on the compound's maximum UV absorbance and verification with standards, the compound was identified as p-coumaric acid.

[0068] (6) The process of analyzing the structure of the compound in the eluent with a retention time of 46.05 min is as follows:

[0069] The primary mass spectrum of compound 6 shows m / z 279.1125 and m / z 559.1551, corresponding to [MH], respectively. - [2M-H] -The molecular weight of the compound was determined to be 280. The compound exhibits a maximum UV absorption peak at 264 nm, confirming it as safflower bean acid.

[0070] (7) The structural analysis of the compound in the eluent with a retention time of 48.09 min is as follows; see the specific primary mass spectrum. Figure 1 The secondary mass spectrum is shown below. Figure 2 :

[0071] The primary mass spectrum of compound 7 shows m / z 651.1922 and m / z 627.1910, which correspond to [M+Na], respectively. + and [MH] - The molecular formula of the compound was determined to be C. 28 H 36 O 16 The molecular weight is 628. After collecting the fraction of this compound by HPLC, nuclear magnetic resonance analysis confirmed that the compound is 1-O-gentiopicroside-5-hydroxy-3,7-diene-[2,6]cyclopentanopyranyl-3-methyl ester. Since this compound was discovered for the first time and is found only in Vitex honey, this invention names it Vitexin and uses it as a marker component of Vitex honey. The nuclear magnetic resonance spectrum of this compound is shown below. Figure 4 As shown (top image is proton spectrum, bottom image is carbon spectrum), the secondary mass spectrometry fragmentation pathway is as follows: Figure 5 The structural formula, 1H NMR spectrum, and 1C NMR spectrum of the compound are as follows:

[0072] Structural formula:

[0073]

[0074] Hydrogen spectrum:

[0075] 1H NMR (600 MHz, CD3OD) δ 7.94–7.90 (m, 2H, 13, 17), 6.86–6.82 (m, 2H, 14, 16), 6.34 (dd, J=6.1, 2.0 Hz, 1H, 3), 5.90 (p, J=1.7 Hz, 1H, 7), 5.12 (dd, J=6.1, 4.0 Hz, 1H, 4), 5.04 (dt, J=14.8, 1.9 Hz, 1H, 10′), 4.99 (d, J=7.7 Hz, 1H, 1), 4.94 (dt, J=15.1, 1.9 Hz, 1H, 10”), 4.68 (d, J=7.9 Hz, 1H, 1′), 4.57 (dp, J=5.9, 2.0 Hz, 1H, 6), 4.35 (d, J=7.8 Hz, 1H, 1”), 4.09 (dd, J=12.1, 2.0 Hz, 1H, 6”), 3.77 (dd, J=11.9, 2.4 Hz, 1H, 6”′), 3.74 (dd, J=12.1, 6.9 Hz, 1H, 6”′), 3.61 (dd, J=11.9, 5.4 Hz, 1H, 6””), 3.48 (ddd, J=9.2, 6.9, 2.0 Hz, 1H, 5′), 3.36 (t, J=9.1 Hz, 1H, 3′), 5.92–5.88 (m, 0H), 3.33–3.26 (m, 6H, 4′, 3”, 4”), 3.25 (dd, J=9.2, 7.9 Hz, 1H, 2′), 3.19 (ddd, J=9.1, 5.4, 2.3 Hz, 1H, 5”), 3.16 (dd, J=8.8, 7.7 Hz, 1H, 2”), 2.95 (t, J=7.8 Hz, 1H, 9), 2.68 (dddd, J=7.9, 5.9, 3.9, 2.0 Hz, 1H, 5).

[0076] Carbon spectrum:

[0077] 13C NMR (151 MHz, CD3OD) δ 167.70 (11), 163.61 (15), 142.68, 141.84 (3), 132.95 (13, 17), 132.92 (7), 122.19 (12), 116.27 (14, 16), 105.42 (4), 105.12 (1”), 100.28 (1′), 98.22 (1), 82.82 (6), 77.95 (3”), 77.89 (3′), 77.79 (5”), 77.68 (5′), 75.14 (2”), 74.85 (2′), 71.72 (4”), 71.39 (4′), 69.98 (6′), 63.74 (10), 62.59 (6”), 48.56 (9), 46.53 (5).

[0078] (8) The process of analyzing the structure of the compound in the eluent with a retention time of 60.79 min is as follows:

[0079] The primary mass spectrum of compound 8 shows m / z 247.1294, m / z 287.1211, m / z 551.2592, and m / z 263.1332, which correspond to [M-H2O], respectively. + [M+Na] + [2M+Na] + The molecular weight of the compound was determined to be 264 based on the presence of [MH]-. By adding standards and comparing the retention time, maximum absorption wavelength, and secondary characteristic ion fragments, the compound was identified as trans-trans abscisic acid.

[0080] (9) The process of structural analysis of the compound in the eluent with a retention time of 69.32 min is as follows:

[0081] The primary mass spectrum of compound 9 shows m / z 247.1246, m / z 287.116, m / z 551.2556, and m / z 263.4238, which correspond to [M-H2O], respectively. + [M+Na] + [2M+Na] + and [MH] - The molecular weight of the compound was determined to be 264. By adding standards and comparing the retention time, maximum absorption wavelength, and secondary characteristic ion fragments, the compound was identified as cis-trans abscisic acid.

[0082] (10) The process of analyzing the structure of the compound in the eluent with a retention time of 78.99 min is as follows:

[0083] The primary mass spectrum of compound 10 shows m / z 287.0918, m / z 309.0940, m / z 595.1577, and m / z 285.4403, corresponding to [M+H], respectively. + [M+Na] + [2M+Na] + and [MH] - The molecular formula of the compound was determined to be C. 16 H 14 O5, with a molecular weight of 286, was identified as 5-methoxybryophyll by combining the secondary mass spectrum, maximum UV absorption, and verification with standards.

[0084] (11) The process of analyzing the structure of the compound in the eluent with a retention time of 83.71 min is as follows:

[0085] The primary mass spectrum of compound 11 shows m / z 273.0760, m / z 295.0587, and m / z 271.4058, corresponding to [M+H], respectively. + [M+Na] + and [MH] - The molecular formula of the compound was determined to be C. 15 H 12 O5, with a molecular weight of 272. It was identified as pinoin by combining the secondary mass spectrum, maximum UV absorption, and verification with standards of compound 11.

[0086] (12) The process of analyzing the structure of the compound in the eluent with a retention time of 104.93 min is as follows:

[0087] The primary mass spectrum of compound 12 shows m / z 287.0679 and m / z 285.0273, corresponding to [M+H], respectively. + and [MH] - The molecular formula of the compound was determined to be C. 15 H 10 O6, with a molecular weight of 286, was identified as kaempferol by combining the secondary mass spectrum of compound 12, its maximum UV absorption, and verification with standards.

[0088] (13) The process of analyzing the structure of the compound in the eluent with a retention time of 114.03 min is as follows:

[0089] The primary mass spectrum of compound 13 shows m / z 287.0820 and m / z 255.3871, corresponding to [M+H], respectively. + and [MH] - The molecular formula of the compound was determined to be C. 15 H 12 O4, with a molecular weight of 286. It was identified as pinocembrin by combining the secondary mass spectrum of compound 13, its maximum UV absorption, and verification with standards.

[0090] (14) The process of structural analysis of the compound in the eluent with a retention time of 116.63 min is as follows:

[0091] The primary mass spectrum of compound 14 shows m / z 306.0792 and m / z 283.4225, corresponding to [M+Na], respectively. + and [MH] - The molecular formula of the compound was determined to be C. 16 H 14 O4, with a molecular weight of 284, was identified as benzyl caffeate by combining the secondary mass spectrum, maximum UV absorption, and verification with standards.

[0092] (15) The process of analyzing the structure of the compound in the eluent with a retention time of 120.81 min is as follows:

[0093] The primary mass spectrum of compound 15 shows m / z 255.0655 and m / z 253.3677, corresponding to [M+H], respectively. + and [MH] - The molecular formula of the compound was determined to be C. 15 H 10 O4, with a molecular weight of 254. It was identified as a coenzyme by combining the secondary mass spectrum of compound 15, its maximum UV absorption, and verification with standards.

[0094] (16) The process of analyzing the structure of the compound in the eluent with a retention time of 124.17 min is as follows:

[0095] The primary mass spectrum of compound 16 shows m / z 271.0603 and m / z 269.3858, corresponding to [M+Na], respectively. + and [MH] - The molecular formula of the compound was determined to be C. 15 H 10 O5, with a molecular weight of 270, was identified as galangin by combining the secondary mass spectrum, maximum UV absorption, and verification with standards of compound 16.

[0096] Example 2

[0097] In this embodiment, the high performance liquid chromatography method of Example 1 was used to detect Vitex honey from fifteen apiaries, and the components of these Vitex honeys were quantitatively analyzed by external standard method.

[0098] Specific information on the source of the honey is shown in Table 1 below:

[0099] Table 1. Honey made from Vitex negundo raw materials

[0100] sample name source Collection time Sample 1 Vitex honey (Vitex negundo Linn.) Hebei 2021.7.6 Sample 2 Vitex honey (Vitex negundo Linn.) Hebei 2021.7.6 Sample 3 Vitex honey (Vitex negundo Linn.) Henan 2021.7.6 Sample 4 Vitex honey (Vitex negundo Linn.) Henan 2021.7.2 Sample 5 Vitex honey (Vitex negundo Linn.) Henan 2021.7.2 Sample 6 Vitex honey (Vitex negundo Linn.) Shanxi 2021.7.2 Sample 7 Vitex honey (Vitex negundo Linn.) Shanxi 2021.7.9 Sample 8 Vitex honey (Vitex negundo Linn.) Shanxi 2021.7.9 Sample 9 Vitex honey (Vitex negundo Linn.) Shaanxi 2021.7.10 Sample 10 Vitex honey (Vitex negundo Linn.) Shaanxi 2021.7.10 Sample 11 Vitex honey (Vitex negundo Linn.) Shaanxi 2021.7.15 Sample 12 Vitex honey (Vitex negundo Linn.) Beijing 2021.7.15 Sample 13 Vitex honey (Vitex negundo Linn.) Beijing 2021.7.16 Sample 14 Vitex honey (Vitex negundo Linn.) Liaoning 2021.7.18 Sample 15 Vitex honey (Vitex negundo Linn.) Liaoning 2021.7.18

[0101] The overlay chromatograms of fifteen different vitex honey samples are shown below. Figure 7 .

[0102] Among the fifteen honey samples, the average content of neochlorogenic acid was 0.3±0.04 mg / kg, the average content of 4-hydroxybenzoic acid was 2.84±0.14 mg / kg, the average content of chlorogenic acid was 0.63±0.08 mg / kg, the average content of caffeic acid was 1.14±0.18 mg / kg, the average content of p-coumaric acid was 0.47±0.12 mg / kg, the average content of safflower-like gentian acid was 1.15±0.23 mg / kg, the average content of vitexin was 141±1.23 mg / kg, and the average content of trans- and trans-abscisic acid was 0.41±0.15 mg / kg. The average content of cis- and trans-abscisic acid was 0.97 ± 0.12 mg / kg, the average content of 5-methoxypinene was 0.49 ± 0.20 mg / kg, the average content of pinene was 0.65 ± 0.32 mg / kg, the average content of kaempferol was 0.23 ± 0.11 mg / kg, the average content of pinocembrin was 0.3 ± 0.02 mg / kg, the average content of benzyl caffeate was 1.21 ± 0.29 mg / kg, the average content of coine was 1.1 ± 0.17 mg / kg, and the average content of galangin was 0.53 ± 0.04 mg / kg. Detailed results are shown in Tables 2 and 3.

[0103] Table 2. Results of compound content determination in Vitex honey (mg / kg)

[0104]

[0105]

[0106] Table 3. Results of compound content determination in Vitex honey (mg / kg)

[0107]

[0108]

[0109] Example 3

[0110] This embodiment uses the high-performance liquid chromatography (HPLC) method of Example 1 to analyze the components of acacia honey, linden honey, rapeseed honey, gallnut honey, Schefflera arborescens honey, safflower honey, wolfberry honey, and vine honey (specific raw material honey source information is shown in Table 4). The HPLC chromatograms of the above honeys are compared with those of Vitex honey from Example 1. The results show that neochlorogenic acid, 4-hydroxybenzoic acid, chlorogenic acid, caffeic acid, p-coumaric acid, safflower gentian acid, vitexin, trans-, trans-abscisic acid, cis-, trans-abscisic acid, 5-methoxypinene, pinoresin, kaempferol, pinosin, benzyl caffeate, caine, and galangin were not simultaneously detected in any of the other monofloral honeys besides Vitex honey. Specific detection results are shown in Table 5.

[0111] Table 4 Information on Other Raw Honey Sources

[0112]

[0113]

[0114] Table 5 Information on whether other honeys contain sixteen marker compounds.

[0115]

[0116]

[0117]

[0118]

[0119] Example 4

[0120] This embodiment uses the high-performance liquid chromatography (HPLC) method described in Example 1 to analyze seven commercially available samples of Vitex honey (from different brands on different sales platforms). Analysis of the HPLC chromatograms revealed that the HPLC chromatograms of four brands of Vitex honey were consistent with the standard fingerprint chromatogram, and the content of vitexin was higher than 43.2 mg / kg. The contents of other components were close to those in the raw honey samples. Therefore, these four brands were identified as high-quality Vitex honey. Two brands of Vitex honey showed abnormal peaks in their chromatograms, significantly different from the standard fingerprint chromatogram, and the content of the marker component was lower than 43.2 mg / kg. Therefore, this invention suggests that these two brands were adulterated honey. In one brand, although the 16 identified components were detected, abnormal peaks were still present. Analysis and verification by this invention confirmed that the abnormal peak was methyl eugenol. Therefore, this invention suggests that this brand of honey was mixed with rapeseed honey.

[0121] This invention compares and analyzes the characteristic components of Vitex honey with those of acacia honey, linden honey, rapeseed honey, gallnut honey, Schefflera honey, safflower honey, wolfberry honey, and vine honey. None of these components were simultaneously detected in other monofloral honeys besides Vitex honey. The analysis revealed the absence of neochlorogenic acid, 4-hydroxybenzoic acid, chlorogenic acid, caffeic acid, p-coumaric acid, safflower benzyl acid, vitexin, trans-, trans-abscisic acid, cis-, trans-abscisic acid, 5-methoxypinene, pinoresin, kaempferol, pinoylsin, benzyl caffeate, caine, and galangin. Therefore, this invention uses these 16 components as characteristic plant compound markers for Vitex honey and applies them to the construction of a system for evaluating the authenticity and quality of Vitex honey.

[0122] 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. A vitexin, characterized in that, It has the structure shown in Equation 1: Formula 1.

2. The method for preparing vitexin according to claim 1, characterized in that, Characteristic components in honey, including Vitex honey, were separated by high-performance liquid chromatography (HPLC). During HPLC separation, mobile phase A was a 0.18–0.22% aqueous acetic acid solution, and mobile phase B was a 0.18–0.22% methanolic acetic acid solution. The chromatographic column was a Phenomenex Gemini C18 column, with a flow rate of 0.6–0.8 mL / min, an injection volume of 15–25 μL, a column temperature of 34–36℃, and a detection wavelength of 270 ± 10 nm. Gradient elution was employed. The gradient elution program was as follows: From 0 to 10 minutes, the percentage of mobile phase B increased from 9% to 14%. Over 10–18 minutes, the percentage of mobile phase B increased from 14% to 15%. Over 18–28 minutes, the percentage of mobile phase B increased from 15% to 17%. Over 28–40 minutes, the percentage of mobile phase B increased from 17% to 26%. Over 40–44 minutes, the percentage of mobile phase B increased from 26% to 33%. From 44 to 54 minutes, the percentage of mobile phase B increased from 33% to 34%. From 54 to 64 minutes, the percentage of mobile phase B increased from 34% to 35%. From 64 to 69 minutes, the percentage of mobile phase B increased from 35% to 37%. From 69 to 74 minutes, the percentage of mobile phase B increased from 37% to 38%. 74-84 min, mobile phase B maintained 38% to 41%; At 84-89 min, the percentage of mobile phase B increased from 41% to 44%. From 89 to 96 minutes, the content of mobile phase B increased from 44% to 54%. From 96 to 126 minutes, the content of mobile phase B increased from 54% to 65%. Between 126 and 135 minutes, the content of mobile phase B increased from 65% to 80%. The sum of the volumes of mobile phase A and mobile phase B is 100%. The high-performance liquid chromatography method further includes a step of extracting characteristic components from honey using a solid-phase extraction column before high-performance liquid chromatography separation; a honey pretreatment step is also included before extracting characteristic components from honey using a solid-phase extraction column; after separating characteristic components from honey by high-performance liquid chromatography, the target peak eluent is collected, and the retention time of the target peak is 48.09 min.

3. The method for preparing vitexin according to claim 2, characterized in that, In the step of extracting characteristic components from honey using a solid-phase extraction column, a Strata-XA solid-phase extraction column is used for extraction, with methanol as the activator, water as the balancing agent and eluent, and formic acid methanol solution as the eluent. The obtained eluent is dried, reconstituted with methanol, and then filtered.

4. The method for preparing vitexin according to claim 3, characterized in that, The pore size of the filter membrane during filtration is 0.20~0.25μm; the eluent is a formic acid methanol solution with a concentration of 8~15%.

5. The method for preparing vitexin according to any one of claims 2-4, characterized in that, The honey pretreatment includes the following steps: after mixing honey with water, adjust the pH value to 6.5~7, then centrifuge at 8800~9200 rpm for 18~22 min, and take the supernatant.

6. The application of vitexin as described in claim 1 or vitexin prepared by any one of claims 2-5 in the identification of vitex honey.

7. A method for identifying Vitex honey, characterized in that, This includes detecting characteristic components in the honey using high-performance liquid chromatography (HPLC). When the characteristic components contain vitexin, or simultaneously contain vitexin and neochlorogenic acid, 4-hydroxybenzoic acid, chlorogenic acid, caffeic acid, etc. p - Coumaric acid, safflower coumaric acid, trans, trans-abscisic acid, cis, trans-abscisic acid, 5-methoxypinene, pinene, kaempferol, pinosin, benzyl caffeate, caine, galangin, then the honey to be tested is determined to be Vitex honey; the vitexin is as described in claim 1; the high performance liquid chromatography method is the high performance liquid chromatography method in the preparation method of vitexin according to any one of claims 2-5.

8. The method for identifying Vitex honey according to claim 7, characterized in that, When the content of neochlorogenic acid in the honey to be tested is 0.12~0.61 mg / kg, the content of 4-hydroxybenzoic acid is 0.91~8.27 mg / kg, the content of chlorogenic acid is 0.21~1.83 mg / kg, and the content of caffeic acid is 0.14~1.86 mg / kg, p The content of coumaric acid is 0.16~0.97 mg / kg, the content of safflower coumaric acid is 0.19~4.04 mg / kg, the content of vitexin is 43.2~268.76 mg / kg, the content of trans- and trans-abscisic acid is 0.18~0.99 mg / kg, the content of cis- and trans-abscisic acid is 0.14~2.56 mg / kg, the content of 5-methoxypinene is 0.34~0.76 mg / kg, the content of pinene is 0.18~1.56 mg / kg, the content of kaempferol is 0.13~0.45 mg / kg, the content of pinocembrin is 0.10~0.69 mg / kg, the content of benzyl caffeate is 0.75~2.07 mg / kg, the content of caine is 0.52~2.04 mg / kg, and the content of galangin is 0.29~1.38 mg / kg. If the concentration is mg / kg, then the honey to be tested is determined to be Vitex honey.

9. A fingerprint spectrum of Vitex honey, characterized in that, It is prepared by processing Vitex honey using the high-performance liquid chromatography method described in any one of claims 2-5.

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