CN113248417B - Amino acid p-benzoquinone compound and application thereof as characteristic marker of black honey of rice-pudding - Google Patents
Amino acid p-benzoquinone compound and application thereof as characteristic marker of black honey of rice-pudding Download PDFInfo
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Abstract
The invention relates to the technical field of food detection, in particular to an amino acid p-benzoquinone compound and application thereof as a characteristic marker of black honey of rice-flower. The invention discovers the color-forming compound in the black honey of the rice-roll flower for the first time. The compound can be used as a characteristic marker compound of the black rice flower honey for identifying the black rice flower honey. The method for identifying the black honey of the rice-roll flower has high specificity and sensitivity, and the detection limit of the characteristic marker compound can reach 10 mug/kg. The identification method has the characteristics of simple operation, high efficiency and the like, is convenient to popularize and apply, and has important significance for identification and authenticity evaluation of the black honey of the rice cluster flowers.
Description
Technical Field
The invention relates to the technical field of food detection, in particular to an amino acid p-benzoquinone compound and application thereof as a characteristic marker of black honey of rice-flower.
Background
The Leucoscepta Miquel is a plant belonging to Leucoscepta of Labiatae, and is mainly distributed in southeast of Yunnan province, Sichuan province and Tibet. Leucoscepta is a rare tall shrub or arbor in the family Labiatae, up to 15 meters. The flower period of the rice ball flower is long, and the flower honey can be collected and brewed by bees to obtain rare black honey. The roots and leaves of the rice-roll flower have medicinal values, and the black honey of the rice-roll flower is derived from the medicinal plant of the rice-roll flower and is recently attracted by people as a typical black food. The market value of the black rice dumpling flower honey is high, and the phenomenon of adulteration exists, so that the development of the characteristic marker of the black rice dumpling flower honey and the effective method for identifying the black rice dumpling flower honey are of great significance.
Disclosure of Invention
The invention aims to provide a compound which is a color-developing compound separated from black rice-flower honey and can be used as a characteristic marker of the black rice-flower honey. The invention also aims to provide application of the compound and an identification method of the black honey of the rice-pudding.
According to the invention, through analyzing the color substances of the black honey of the flower of Chinese feverfew, the color substances are found to have strong absorption at 254nm and 370 nm, and further through identifying the color substances by UHPLC-UV-Q-TOF MS, two characteristic ions in the black honey of the flower of Chinese feverfew are obtained, wherein m/z of the characteristic ions is 323.1238 and 351.1551 respectively, so that two compounds in the black honey of the flower of Chinese feverfew and an inferred structural formula of the two compounds are obtained. The two compounds in the black honey of the rice dumpling flower are separated and purified to obtain a pure product, and the structure of the pure product is determined by high-resolution mass spectrum and nuclear magnetism. And further obtaining two pure compounds with the structure by a chemical synthesis method, comparing and verifying the two pure compounds, confirming the structures by high-resolution mass spectrum and nuclear magnetism, and completely conforming the structures of the synthesized compounds and the two pure compounds obtained by separating the black honey of the rice flower. The invention further detects and analyzes a large number of black rice dumpling flower honey samples of different years and different sources and other honey samples such as locust honey, date honey, linden honey and the like through UHPLC-UV-Q-TOF MS, and finds that the two compounds are only contained in the black rice dumpling flower honey, but the other honey does not contain the two compounds, and the contents of the two compounds are stable in different black rice dumpling flower honey samples, so that the black rice dumpling flower honey identification and authenticity evaluation can be carried out by using the two compounds as characteristic markers of the black rice dumpling flower honey.
Specifically, the invention provides the following technical scheme:
firstly, the invention provides a compound, the chemical structural formula of which is shown as formula (I) or formula (II):
formula (I);
formula (II).
The molecular formulas of the compounds shown in the formula (I) and the formula (II) are respectively C15H18N2O6And C17H22N2O6Belongs to amino acid p-benzoquinone compounds.
The compounds shown in the formula (I) and the formula (II) are color-developing compounds separated from black honey of the rice flower. According to UHPLC-UV-Q-TOF MS detection, the chromatogram of the compound shown in the formula (I) should contain m/z 323.1238 and the error of the accurate mass number should be less than or equal to 5ppm, and the chromatogram of the compound shown in the formula (II) should contain m/z 351.1551 and the error of the accurate mass number should be less than or equal to 5 ppm.
The compound can be obtained by separating and purifying black honey of the rice cluster flower, and the specific separation and purification method is as follows:
mixing flos Micheliae Championii Mel with water, removing sugar with macroporous resin, filtering, performing gradient elution with water (mobile phase A) 85-95% and methanol (mobile phase B) 5-15% by preparing liquid phase, and detecting at 370 nm with flow rate of 20-30 mL/min and column temperature of 20-50 deg.C. Collecting the preparation solution of 6.5-7.8 min, i.e. the compound shown in formula (I), and collecting the preparation solution of 10.2-12.5 min, i.e. the compound shown in formula (II).
The compound can also be prepared by a chemical synthesis method, and the specific preparation method comprises the following steps: reacting the first amino acid, the second amino acid, sodium carbonate and p-benzoquinone in methanol,
wherein the first amino acid is proline, and for the compound of formula (I), the second amino acid is gamma-aminobutyric acid, and for the compound of formula (II), the second amino acid is leucine.
Specifically, a first amino acid, a second amino acid and sodium carbonate in a molar ratio of 2:2:3 are mixed with methanol to obtain a mixture, the mixture is mixed with p-phenylenediamine and then reacts for 1-4 h at 15-30 ℃, and the molar ratio of the mixture to the p-phenylenediamine is 3: 1.
Preferably, in the above preparation method, the molar volume ratio of the amount of the mixture to the amount of methanol is mmol: ml is (0.5-1): 2.
the chemical synthesis method can be used for efficiently preparing the compounds shown in the formulas (I) and (II).
The following methods can be used for purification of the compounds of formula (I) and formula (II): passing the system after the reaction of the chemically synthesized target compound through a 0.45-micrometer filter membrane, performing gradient elution by using the prepared liquid phase, 85-95% of water (mobile phase A) and 5-15% of methanol (mobile phase B) and the like, taking 370 nm as a detection wavelength, controlling the flow rate at 25-35mL/min and the column temperature at 20-50 ℃, and collecting the prepared liquid.
Based on the functions of the compounds of the present invention, the present invention provides the uses thereof as described below.
The invention provides an application of the compound or the compound prepared by the preparation method in serving as a characteristic marker of black honey of the rice-roll flower.
The invention also provides application of the compound or the compound prepared by the preparation method in identifying the black honey of the rice-flower or detecting the authenticity of the black honey of the rice-flower.
The compounds shown in the formula (I) and the formula (II) are only contained in the black rice-pudding honey, and the two compounds cannot be detected in other honey, so that the compounds can be used for distinguishing the black rice-pudding honey from other honey.
The adulterated black rice ball honey is obtained by adding other honey or raw materials except the black rice ball honey, so that the content of the compounds shown in the formulas (I) and (II) is obviously reduced, and therefore, whether the black rice ball honey is adulterated or not can be judged by detecting the content of the compounds shown in the formulas (I) and (II), namely, the authenticity evaluation of the black rice ball honey is carried out.
The invention provides an identification method of black honey containing flos reuteri, which is characterized in that a honey sample is detected by taking a compound as a characteristic marker, if the content of the compound shown in a formula (I) in the honey sample is more than or equal to 0.5mg/kg and/or the content of the compound shown in a formula (II) is more than or equal to 0.3mg/kg, the honey sample is judged to be the black honey containing the flos reuteri, and if the content of the compound shown in the formula (I) in the honey sample is less than 0.5mg/kg and the content of the compound shown in the formula (II) in the honey sample is less than 0.3mg/kg, the honey sample is judged to be other honey except the black honey containing the flos reuteri or the black honey containing the flos reuteri.
The content of the compounds shown in the formula (I) and the formula (II) in a large number of black rice flower honey samples from different sources is detected, and the results show that the content of the compounds shown in the formula (I) and the content of the compounds shown in the formula (II) are respectively stabilized within the range of more than or equal to 0.5mg/kg and more than or equal to 0.3 mg/kg.
Preferably, in the above identification method, if the content of the compound represented by formula (I) in the honey sample is 0.5-31 mg/kg and/or the content of the compound represented by formula (II) is 0.3-12 mg/kg, the honey sample is determined to be black rice-flower honey, and if the content of the compound represented by formula (I) in the honey sample and the content of the compound represented by formula (II) are not within the above range, the honey sample is determined to be other honey except black rice-flower honey or black adulterated black rice-flower honey.
Preferably, when the honey sample is tested by UHPLC-UV-Q-TOF MS, the chromatogram of the compound shown in formula (I) should contain m/z 323.1238 with the error of accurate mass number less than or equal to 5ppm, and the chromatogram of the compound shown in formula (II) should contain m/z 351.1551 with the error of accurate mass number less than or equal to 5 ppm.
Specifically, a honey sample to be detected is detected by adopting UHPLC-DAD-Q-TOF/MS, and when an obtained spectrogram contains a characteristic peak (323.1238 +/-5 ppm) corresponding to the compound shown in the formula (I) and the corresponding content is more than or equal to 0.5mg/kg, and/or when a characteristic peak (351.1551 +/-5 ppm) corresponding to the compound shown in the formula (II) and the corresponding content is more than or equal to 0.3mg/kg, the honey is judged to be black honey of the rice ball flowers.
Preferably, the UHPLC-UV-Q-TOF MS is an ultra-high performance liquid-diode array-quadrupole-time-of-flight mass spectrometer (UHPLC-DAD-Q-TOF/MS).
The content of the compound represented by the formula (I) and the compound represented by the formula (II) can be calculated according to the standard curve and the peak areas of the compounds represented by the formula (I) and the formula (II) detected in the honey sample.
Based on the characteristic peaks in the chromatogram provided above, even in the case of a reference product without the compounds represented by the formulas (I) and (II), whether the honey sample to be tested is the black honey of the rice ball flower can be judged according to whether the characteristic peaks are contained, and the content of the compounds represented by the formulas (I) and (II) can be calculated by integrating the peak areas of the parent ions.
Preferably, the liquid phase conditions for detecting the compounds shown in the formula (I) and the formula (II) by the UHPLC-UV-Q-TOF MS are as follows: using one of a reversed phase chromatographic column C18 and a phenyl column, wherein the mobile phase is as follows: the mobile phase A is 0.08-0.12% formic acid water solution, the mobile phase B is methanol, and the gradient elution procedure is as follows: 0-5 min, 5% mobile phase B; 5-15min, 5-95% of mobile phase B; 15-16 min, 95-5% of mobile phase B, and then running for 4 min; the flow rate is 0.2-0.8 mL/min, the column temperature is 20-50 deg.C, and the ultraviolet detector wavelength is set at 254 + -5 nm and 370 + -5 nm.
The amount of the sample to be detected in the liquid phase is preferably 1 to 5. mu.L.
The above-mentioned liquid phase detection is preferably carried out using an AQ-C18 column (2 μm, 2.1X 50 mm) column.
Preferably, the mass spectrum conditions of UHPLC-UV-Q-TOF MS detection are as follows: electrospray mass spectrometry, wherein the temperature of atomizing gas is 250-.
Mixing the honey sample to be detected with ultrapure water according to the ratio of 1:5 before the UHPLC-UV-Q-TOF MS detection, filtering by using a filter membrane, and then carrying out UHPLC-UV-Q-TOF MS detection.
The invention considers the accuracy and precision of the UHPLC-UV-Q-TOF MS detection method for detecting the compounds shown in the formula (I) and the formula (II), and the result shows that the accuracy of the method is between 85 and 115 percent, the variation coefficients are less than 12 percent, the detection limit can reach 10 mu g/kg, and the method completely meets the requirements of analysis and detection.
The invention has the beneficial effects that: the invention discovers the color generation compounds in the black rice-pudding honey for the first time, and the structural formulas of the color generation compounds are shown as a formula (I) and a formula (II). The two compounds can be used as characteristic marker compounds of the black rice flower honey for identifying the black rice flower honey. The method for identifying the black honey of the rice cluster flowers has high specificity and sensitivity, the detection limit of a target compound can reach 10 mu g/kg, the accuracy is between 85 and 115 percent, and the variation coefficients are all less than 12 percent. The method for identifying the black honey of the rice cluster flowers has the characteristics of simplicity in operation, high efficiency and the like, is convenient to popularize and apply, and has important significance for identification and authenticity evaluation of the black honey of the rice cluster flowers.
Drawings
FIG. 1 is a secondary mass spectrum of a compound represented by the formula (I) in example 1 of the present invention.
FIG. 2 is a secondary mass spectrum of the compound represented by the formula (II) in example 1 of the present invention.
FIG. 3 is a hydrogen spectrum of the compound represented by the formula (I) in example 1 of the present invention.
FIG. 4 is a hydrogen spectrum of the compound represented by the formula (II) in example 1 of the present invention.
FIG. 5 is a chromatogram of a chemical synthesis method according to example 2 of the present invention for preparing a compound represented by formula (I).
FIG. 6 is a chromatogram of a chemical synthesis method for producing a compound represented by formula (II) in example 2 of the present invention.
FIG. 7 is a chromatogram of a compound represented by formula (I) in a honey sample of example 2 of the present invention.
FIG. 8 is a chromatogram of the compound represented by the formula (II) in the honey sample of example 2 of the present invention.
Detailed Description
The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.
Example 1 separation and purification of color-developing Compounds from Black Honey of Oryza Glutinosa
By analyzing the color substances of the black honey of the rice doughnuts, the color substances are found to have strong absorption at 254nm and 370 nm, and the color substances are further identified by ultra high performance liquid chromatography-diode array-quadrupole-time of flight mass spectrometry (UHPLC-DAD-Q-TOF/MS).
The UHPLC-DAD-Q-TOF/MS method is as follows:
mixing black honey of the rice-roll flower with ultrapure water according to the ratio of 1:5, and filtering by using a filter membrane. Using a AQ-C18 column (2 μm, 2.1X 50 mm) chromatography, mobile phases of 0.1% aqueous formic acid (A) and methanol (B), a gradient elution procedure was as follows: 0-5 min, 5% mobile phase B; 5-15min, 5-95% of mobile phase B; 15-16 min, 95-5% of mobile phase B, and then running for 4 min; the flow rate was 0.3 mL/min, the column temperature was 30 ℃ and the amount of sample was 2. mu.L. The UV detector wavelengths were set at 254 + -5 nm and 370 + -5 nm. The mass spectrum detection conditions are as follows: in the electrospray mass spectrometry, the temperature of atomizing gas is 350 ℃, the flow rate of atomizing gas is 12L/min, the pressure of atomizing gas is 45 psi, the temperature of sheath gas is 300 ℃, the flow rate of sheath gas is 13L/min, the ionization voltage is 3.0 kv, the fragment voltage is 125V, the scanning mode is a positive ion mode, and the full scanning and multi-reaction monitoring modes are adopted.
Through the detection, two characteristic ions in the black honey of the rice-roll mass flower are obtained, wherein m/z of the two characteristic ions is 323.1238 and 351.1551 respectively, and ion fragments are shown in the figure 1 and the figure 2 respectively.
According to the ion fragment result, the chemical structural formulas of the two compounds are respectively shown as a formula (I) and a formula (II), and the molecular formulas are respectively C15H18N2O6And C17H22N2O6。
The two compounds in the black honey of the rice cluster flowers are separated and purified to prepare a pure product, and the specific method is as follows: mixing 2 kg of flos Micheliae Championii honey with 6L of deionized water, thoroughly mixing, removing sugar with macroporous resin, filtering with 0.45 μm filter membrane, preparing liquid phase, gradient eluting with 90% water (mobile phase A) and 10% methanol (mobile phase B), and detecting with 370 nm wavelength. The sample introduction volume is 1.0 mL, the flow rate is 25 mL/min, and the column temperature is 30 ℃. Collecting the preparation solution of 6.5-7.8 min, i.e. the compound shown in formula (I), collecting the preparation solution of 10.2-12.5 min, i.e. the compound shown in formula (II), and vacuum freeze drying to obtain pure product.
The structure of the compound is determined to be the structure of the inferred compound through high-resolution mass spectrometry and nuclear magnetism.
The results of nuclear magnetic assays for the compounds of formula (I) and formula (II) are as follows (fig. 3 and 4):
formula (A), (B) andI）：lH NMR ( 600 MHz, DMSO-d6) δ 12.22 ( s, 1H ) , 12.02 ( s, 1H ) , 8.32 ( s, 1H ), 5.01 ( s, 1H ) , 5.00 ( s, 1H), 3.36 ( dd, J=5.4, 3.0 Hz, 1H ), 3.30-3.26 ( m, 2H ), 2.87-2.80 ( m, 2H ), 2.31 ( t, J=7.2 Hz, 2H ), 1.92-1.82 ( m, 4H ), 1.64-1.54 ( m, 2H )。
formula (II):lH NMR ( 600 MHz, DMSO-d6) δ 12.60 ( s, 1H ) , 12.20 ( s, 1H ) , 8.32 ( s, 1H ), 5.01 ( s, 1H ) , 5.00 ( s, 1H), 3.36 ( dd, J=5.4, 3.0 Hz, 1H ), 3.30-3.26 ( m, 1H ), 2.87-2.80 ( m, 2H ), 1.92-1.88 ( m, 1H ), 1.75 ( dd, J=7.2, 6.6 Hz, 2H ), 1.70-1.54 ( m, 3H ), 1.49 ( m, 1H ), 0.94 ( d, J=7.8, 6H )。
example 2 chemical Synthesis of color Forming Compounds in Black Honey from Oryza Glutinosa
Two compounds separated and purified in example 1 are obtained by a chemical synthesis method, and the specific synthesis method is as follows:
a compound (C) represented by the formula (I)15H18N2O6): adding 0.18 mmol of proline, 0.18 mmol of gamma-aminobutyric acid and 0.27 mmol of Na2CO3After mixing with 2 mL of methanol, 0.21mmol of p-phenylenediamine is added and the reaction is carried out for 2 h at 25 ℃.
A compound (C) represented by the formula (II)17H22N2O6): adding 0.18 mmol of proline, 0.18 mmol of leucine and 0.27 mmol of Na2CO3After mixing with 2 mL of methanol, 0.21mmol of p-phenylenediamine is added and the reaction is carried out for 2 h at 25 ℃.
Separating and purifying the synthesized compound to prepare a pure product, wherein the specific method comprises the following steps:
after the reaction for synthesizing the target compound was completed, the resultant was passed through a 0.45 μm filter, and then the resultant liquid phase was subjected to gradient elution with 90% water (mobile phase A) and 10% methanol (mobile phase B), and the detection wavelength was 370 nm. The sample introduction volume is 0.5 mL, the flow rate is 30 mL/min, and the column temperature is 30 ℃. Collecting the preparation solution, and vacuum freeze drying to obtain pure product.
The two color-developing compounds obtained by separating the pure products of the two compounds from the black honey of the rice-roll flower are compared and verified in structure by adopting high-resolution mass spectrometry (figure 1 and figure 2) and nuclear magnetism. The results showed that the two chemically synthesized compounds were completely identical in structure to the two pure compounds obtained in the black honey of rice-flower (fig. 5, 6, 7 and 8).
Example 3 detection method of characteristic marker and evaluation of detection Effect thereof
UHPLC-DAD-Q-TOF/MS is adopted to detect the compounds shown in the formula (I) and the formula (II), and the specific method is as follows:
liquid chromatography detection conditions: using a AQ-C18 column (2 μm, 2.1X 50 mm) chromatography, mobile phases of 0.1% aqueous formic acid (A) and methanol (B), a gradient elution procedure was as follows: 0-5 min, 5% mobile phase B; 5-15min, 5-95% of mobile phase B; 15-16 min, 95-5% of mobile phase B, and then running for 4 min; the flow rate was 0.3 mL/min, the column temperature was 30 ℃ and the amount of sample was 2. mu.L. The UV detector wavelengths were set at 254 + -5 nm and 370 + -5 nm.
The mass spectrum detection conditions are as follows: in the electrospray mass spectrometry, the temperature of atomizing gas is 350 ℃, the flow rate of atomizing gas is 12L/min, the pressure of atomizing gas is 45 psi, the temperature of sheath gas is 300 ℃, the flow rate of sheath gas is 13L/min, the ionization voltage is 3.0 kv, the fragment voltage is 125V, the scanning mode is a positive ion mode, and the full scanning and multi-reaction monitoring modes are adopted.
The effect evaluation of the detection method is specifically as follows:
1. accuracy analysis
The accuracy of the detection method was examined in terms of additive recovery. The test result of the test method shows that the accuracy of the method is between 85 and 115 percent.
2. Precision analysis
The peak areas RSD (n = 6) of the compound I and the compound II are both less than 5% according to the injection analysis (n = 6) of the detection method of 50 mug/L of the solution of the compound I and the solution of the compound II respectively.
3. Stability analysis
Preparing a sample solution according to a method of 1:5 (mass/volume ratio) of a honey sample and deionized water, placing the sample solution at room temperature for 0, 6, 12, 16 and 24 hours respectively, and determining according to the detection method. The results show that the RSD of the process is less than 5% each.
4. Analysis of detection limits
LOD is 3 times the noise signal and LOQ is 10 times the noise signal. The results show that the detection limit of the compounds represented by the formula (I) and the formula (II) can reach 10 mug/kg, and the quantification limit is 33 mug/kg.
Example 4 content detection of characteristic markers of black Leucocephalum Mel in different Mel
1. Sample source: 50 parts of real honey samples of the black honey of the flower of Chinese feverfew are directly collected from bee farms in different areas in different years and different seasons, 20 parts of real honey samples of other honey (including date honey, acacia honey and linden honey) are collected respectively, and the content of the compounds shown in the formula (I) and the formula (II) is detected by using the total 110 parts of honey samples.
2. Sample pretreatment: mixing the honey sample with ultrapure water according to the ratio of 1:5, and filtering by using a filter membrane.
3. UHPLC-DAD-Q-TOF/MS detection
Liquid chromatography detection conditions: using a AQ-C18 column (2 μm, 2.1X 50 mm) chromatography, mobile phases of 0.1% aqueous formic acid (A) and methanol (B), a gradient elution procedure was as follows: 0-5 min, 5% mobile phase B; 5-15min, 5-95% of mobile phase B; 15-16 min, 95-5% of mobile phase B, and then running for 4 min; the flow rate was 0.3 mL/min, the column temperature was 30 ℃ and the amount of sample was 2. mu.L. The UV detector wavelengths were set at 254 + -5 nm and 370 + -5 nm.
The mass spectrum detection conditions are as follows: in the electrospray mass spectrometry, the temperature of atomizing gas is 350 ℃, the flow rate of atomizing gas is 12L/min, the pressure of atomizing gas is 45 psi, the temperature of sheath gas is 300 ℃, the flow rate of sheath gas is 13L/min, the ionization voltage is 3.0 kv, the fragment voltage is 125V, the scanning mode is a positive ion mode, and the full scanning and multi-reaction monitoring modes are adopted.
4. Drawing a standard curve: respectively preparing standard substances of the compounds shown in the formula (I) and the formula (II), performing UHPLC-DAD-Q-TOF/MS detection, drawing a standard curve, obtaining a linear equation of y =360023x +226, wherein x and y respectively represent concentration and peak area, and a correlation coefficient R2Is 0.9995.
The detection result shows that the characteristic peak (323.1238 +/-5 ppm) corresponding to the compound shown in the formula (I) and the characteristic peak (351.1551 +/-5 ppm) corresponding to the compound shown in the formula (II) can be detected in the black honey of the rice cluster flowers. And the characteristic peak (323.1238 +/-5 ppm) corresponding to the compound shown in the formula (I) and the characteristic peak (351.1551 +/-5 ppm) corresponding to the compound shown in the formula (II) are not detected in all the acacia honey, jujube honey and linden honey samples.
The content detection results of the compounds shown in the formula (I) and the formula (II) in each black rice flower honey sample are shown in the table 1 and the table 2.
TABLE 1 determination of the content of the respective target compounds in honey (1)
TABLE 2 determination of the content of the respective target compounds in honey (2)
According to the results, the content ranges of the compounds shown in the formula (I) and the formula (II) in the black rice-pudding honey are respectively more than or equal to 0.5mg/kg and more than or equal to 0.3mg/kg, and the compounds shown in the formula (I) and the formula (II) can not be detected in other honey.
Example 5 identification of characteristic markers for commercial Black Honey of Oryza sativa
Sample source: 60 honey samples were purchased from the market, labeled as various grades and brands of black rice-flower honey.
Sample detection was performed using the UHPLC-DAD-Q-TOF/MS detection method of example 3. The results are shown in table 3, and it was found that the pass rate of the sampled samples was 90% by the method for discriminating rice-ball honey of example 3.
TABLE 3 detection results of commercial Leucas zamiifolia honey
Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Claims (10)
1. A compound having a chemical structure according to formula (I) or formula (II):
2. A process for preparing a compound of claim 1, comprising: reacting the first amino acid, the second amino acid, sodium carbonate and p-benzoquinone in methanol,
the first amino acid is proline, and the second amino acid is gamma-aminobutyric acid for the compound shown in the formula (I), and leucine for the compound shown in the formula (II).
3. The preparation method according to claim 2, characterized in that the first amino acid, the second amino acid and sodium carbonate in a molar ratio of 2:2:3 are mixed with methanol to obtain a mixture, the mixture is mixed with p-phenylenediamine and then reacted for 1-4 h at 15-30 ℃, and the molar ratio of the mixture to the p-phenylenediamine is 3: 1.
4. The method according to claim 3, wherein the molar volume ratio of the amount of the mixture to the amount of the methanol is mmol: ml is (0.5-1): 2.
5. use of the compound of claim 1 or the compound prepared by the preparation method of any one of claims 2 to 4 as a characteristic marker of black rice flower honey.
6. Use of the compound of claim 1 or the compound prepared by the preparation method of any one of claims 2 to 4 for identifying or detecting the authenticity of black honey of rice flower.
7. The identification method of the black honey containing the flos reuteri is characterized in that a honey sample is detected by taking the compound as the characteristic marker in claim 1, if the content of the compound shown in the formula (I) in the honey sample is more than or equal to 0.5mg/kg and/or the content of the compound shown in the formula (II) is more than or equal to 0.3mg/kg, the honey sample is judged to be the black honey containing the flos reuteri, and if the content of the compound shown in the formula (I) in the honey sample is less than 0.5mg/kg and the content of the compound shown in the formula (II) in the honey sample is less than 0.3mg/kg, the honey sample is judged to be other honey except the black honey containing the flos reuteri or the black honey containing the flos reuteri.
8. Identification method according to claim 7, characterized in that said honey sample is tested by UHPLC-UV-Q-TOF MS and the chromatogram of the compound of formula (I) contains m/z 323.1238 with an error of the exact mass number of less than or equal to 5ppm and the chromatogram of the compound of formula (II) contains m/z 351.1551 with an error of the exact mass number of less than or equal to 5 ppm.
9. The identification method according to claim 8, wherein the liquid phase conditions detected by UHPLC-UV-Q-TOF MS are as follows: using one of a reversed phase chromatographic column C18 and a phenyl column, wherein the mobile phase is as follows: the mobile phase A is 0.08-0.12% formic acid water solution, the mobile phase B is methanol, and the gradient elution procedure is as follows: 0-5 min, 5% mobile phase B; 5-15min, 5-95% of mobile phase B; 15-16 min, 95-5% of mobile phase B, and then running for 4 min; the flow rate is 0.2-0.8 mL/min, the column temperature is 20-50 deg.C, and the ultraviolet detector wavelength is set at 254 + -5 nm and 370 + -5 nm.
10. The identification method according to claim 9, wherein the mass spectrometric conditions for UHPLC-UV-Q-TOF MS detection are: electrospray mass spectrometry, wherein the temperature of atomizing gas is 250-.
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