Preparation and content determination method of 2, 5-deoxyfructosazine and 2, 6-deoxyfructosazine

Abstract

The invention discloses a method for extracting, separating and purifying 2, 5-deoxyfructosazine (2, 5-DOF) and 2, 6-deoxyfructosazine (2, 6-DOF) monomers from hovenia dulcis thunb and measuring the content of the monomers. The preparation method comprises the following steps: crushing hovenia dulcis thunb, performing reflux extraction with water, filtering, adsorbing filtrate through macroporous adsorption resin, eluting with alkaline water, eluting with an alcohol solution, and recovering a solvent from alcohol eluent under reduced pressure to obtain an extract. And carrying out silica gel column chromatography and preparative high performance liquid chromatography separation and purification on the extract to obtain 2, 5-DOF and 2, 6-DOF monomers. According to the method, the content of 2, 5-DOF and 2, 6-DOF in hovenia dulcis thunb is determined through a solid phase extraction-high performance liquid chromatography-ultraviolet detector (SPE-HPLC-UV) method, LK1300S type macroporous adsorption resin is selected as an SPE stationary phase, an amino chromatographic column is selected as a chromatographic column, an acetonitrile-water mixed solution is adopted as a mobile phase, and the detection wavelength is 275 nm. The preparation method can be used for preparing high-purity 2, 5-DOF and 2, 6-DOF from the hovenia dulcis thunb, and the content determination method can be used for quality control of the hovenia dulcis thunb.

Description

Technical Field

[0001] The invention relates to a method for extracting and separating 2,5-DOF and 2,6-DOF monomers from Hovenia dulcis, and simultaneously determining the contents of the two compounds in Hovenia dulcis by using a SPE-HPLC-UV method, and belongs to the research field of natural medicinal chemistry. Background Art

[0002] Hovenia dulcis is the dried mature seed of the plant Hovenia thunb of the Rhamnaceae family. It is also known as wood honey, chicken feet, jujube, and sour jujube. It is mainly distributed in China, India, North Korea, and Japan. In my country, it is mainly produced in the Qinba Mountain area of ​​Shaanxi, and is also distributed in Sichuan, Hubei, Zhejiang, Jiangsu, Anhui, Fujian, and other places. At present, there are three species of Hovenia and two varieties found in the world, namely Hovenia acerba Lindl., Hovenia dulcis Thunnb., Hovenia trichocarpa Chun et Tsiang., and two varieties, Hovenia trichocarpa var. robusta and Hovenia acerba varkiukiangensis.

[0003] Hovenia dulcis is a traditional Chinese medicine that is both a medicine and a food. It is mild in nature and sweet in taste. It has the effects of quenching thirst, relieving restlessness, detoxifying alcohol, calming nerves, and diuresis. It is often used to relieve symptoms such as drunkenness, fever, thirst, and constipation. Modern pharmacological studies have shown that Hovenia dulcis has the effects of relieving alcohol, protecting the liver, anti-oxidation, anti-tumor, and lowering uric acid.

[0004] The chemical components of Hovenia dulcis plants include flavonoids, terpenes, fatty acids, lignans, alkaloids and other components, among which flavonoids and terpenes are the main chemical components of Hovenia dulcis plants.

[0005] 2,5-deoxyfructosazine (2,5-DOF) and 2,6-deoxyfructosazine (2,6-DOF) monomers are intermediate products of the Maillard reaction and are important flavor precursors. They are closely related to the flavor of food and tobacco during baking and are mainly found in plants such as tobacco. The determination of their content is mainly focused on the determination of 2,5-DOF and 2,6-DOF in glucosamine hydrochloride tablets and tobacco. Currently, the most commonly used methods for detecting this substance are high performance liquid chromatography, ion exchange chromatography tandem mass spectrometry, and gas chromatography. The advantage of these detection methods is that the sample pretreatment is relatively simple, but due to the high polarity and strong water solubility of DOF molecules, the separation effect of high performance liquid chromatography is often poor, especially for complex systems with many interfering substances such as Hovenia dulcis, the target analyte is not easy to be completely separated and therefore difficult to accurately quantify. In addition, since DOF is a non-volatile substance, it needs to be derivatized before gas chromatography analysis.

[0006] So far, there is no literature report on the isolation of 2,5-DOF and 2,6-DOF from Hovenia dulcis and their content determination. Summary of the invention

[0007] The present invention extracts and separates 2,5-DOF and 2,6-DOF monomers from Hovenia dulcis for the first time, and on this basis further invents a new method for simultaneously determining the contents of 2,5-DOF and 2,6-DOF in Hovenia dulcis by using SPE-HPLC-UV.

[0008] The specific technical solutions and research results of the present invention are as follows:

[0009] 1. Extraction, separation and structural identification of 2,5-DOF and 2,6-DOF from Hovenia dulcis

[0010] 10 kg of Hovenia dulcis fruit was taken, crushed, and extracted by reflux with water for 3 times, with 100 L of water added each time. The filtrate was filtered, the filtrate was combined, and adsorbed on a D101 macroporous adsorption resin. It was first eluted with a 0.2% sodium hydroxide aqueous solution, then eluted with water until the effluent was close to neutral, and then eluted with an 85% ethanol solution. The ethanol eluate was decompressed and the solvent was recovered to obtain an extract. The extract was subjected to a first silica gel column chromatography with a mixed solution of ethyl acetate-ethanol-water in a volume ratio of 60:2:1; the component containing 2,5-DOF and 2,6-DOF was subjected to a second silica gel column chromatography with a mixed solution of ethyl acetate-ethanol-water in a volume ratio of 5:4:0.4 as the mobile phase; the component containing 2 The components of 2,5-DOF and 2,6-DOF were subjected to a third silica gel column chromatography using a mixed solution of dichloromethane-methanol-water in a volume ratio of 76:8:1; the components containing 2,5-DOF and 2,6-DOF were subjected to a fourth silica gel column chromatography using a mixed solution of dichloromethane-methanol-water in a volume ratio of 15:3:0.4 as the mobile phase; the components containing 2,5-DOF and 2,6-DOF were finally separated and purified by preparative high performance liquid chromatography using an amino chromatographic column with acetonitrile-water (89:11, V / V) as the mobile phase to obtain 64.2 mg of 2,5-DOF monomer compound and 40.7 mg of 2,6-DOF monomer compound. The obtained monomer compounds were detected by HPLC-UV (HPLC chromatographic conditions: chromatograph: Huapu S6000; chromatographic column: Unitary NH2 (5μm, 100A, 4.6×250mm); mobile phase: acetonitrile-water (89:11, V / V), flow rate: 1.0mL / min; column temperature: 30℃; injection volume: 20μL), and their purities were 95.4% and 93.3%, respectively. By comparing the NMR data of the isolated monomer compounds with the 2,5-DOF and 2,6-DOF NMR data reported in the literature, it was finally determined that they were 2,5-DOF and 2,6-DOF, respectively.

[0011] Table 1 13CNMR (150MHZ) data of 2,5-DOF (δ, ppm, DMSO)

[0012]

[0013] Table 2 2,6-DOF 13 C NMR (150MHZ) data (δ, ppm, DMSO)

[0014]

[0015] 2. Determination of 2,5-DOF and 2,6-DOF in Hovenia dulcis

[0016] 2.1 Chromatographic conditions

[0017] The chromatographic column is Unitary NH2 (5μm, 100A, 4.6×250mm) amino column, the mobile phase is acetonitrile-water (89:11, V / V), the flow rate is 1.0mL / min; the column temperature is 30℃; the detection wavelength is 275nm. (Under this condition, the HPLC chromatograms of the standard and sample are shown in the attached Figure 7 , Attachment Figure 8 )

[0018] 2.2 Preparation of mixed standard stock solution

[0019] Accurately weigh 10.1 mg and 10.2 mg of standard samples of 2,5-DOF (purity of 95.4%) and 2,6-DOF (purity of 93.3%), respectively. Add a small amount of 75% methanol solution and sonicate until completely dissolved. Transfer the solution to a 10 mL volumetric flask, make up to volume with 75% methanol solution and shake well to obtain mixed standard stock solutions with concentrations of 0.964 mg.mL-1 and 0.952 mg.mL-1 for 2,5-DOF and 2,6-DOF, respectively.

[0020] 2.3 Preparation of sample solution

[0021] Take 5g of Hovenia dulcis powder and reflux extract it 3 times. The extraction solvent is pure water. The extraction time is 2h, 1h, and 1h respectively. The amount of pure water is 50mL. The three extracts are combined and filtered. Adsorbed by a 30gLK1300S macroporous adsorption resin column, eluted with 0.2% NaOH solution, rinsed with water to neutrality, desorbed with 60% ethanol, collected the eluate, concentrated to dryness under reduced pressure, dissolved with an appropriate amount of 75% methanol solution by ultrasound, and then diluted to a 2mL volumetric flask. Shake well to obtain the sample solution for later use.

[0022] 2.4 Methodological Review

[0023] 2.4.1 Linear Relationship

[0024] Accurately pipette 5.00mL, 2.00mL, 1.00mL, 0.50mL, 0.20mL, and 0.1mL of the mixed standard stock solution prepared in "2.2" into a 10.0mL volumetric flask, dilute and fix the volume with 75% methanol solution, and shake well to obtain a series of mixed standard solutions, in which the concentrations of 2,5-DOF and 2,6-DOF are: 2,5-DOF: 0.964, 0.482, 0.193, 0.096, 0.048, 0.01 9, 0.001mg.mL-1; the concentrations of 2,6-DOF are: 0.952, 0.476, 0.190, 0.095, 0.048, 0.019, 0.001mg.mL-1, respectively. Before injection, the samples were filtered through a 0.22μm microporous membrane, and then 20μL of the filtrate of each concentration of the standard solution was accurately transferred with a microinjection needle and injected into the HPLC chromatograph. Each sample was injected twice in parallel, and the chromatographic peak areas of 2,5-DOF and 2,6-DOF were recorded and their average values ​​were taken. With the injection mass (m) as the horizontal axis and the peak area (S) as the vertical axis, the standard curve was drawn and the regression equation was calculated. The data are shown in Tables 3 and 4. The experimental results show that the linear range of compound 2,5-DOF is 0.19-19.08μg, and the linear range of 2,6-DOF is 0.19-18.66μg.

[0025] Injection mass (m) = standard solution concentration x injection volume.

[0026] Table 3 Linear relationship investigation data of compound 2,5-DOF

[0027]

[0028] Table 4 Linear relationship investigation data of compound 2,6-DOF

[0029]

[0030] Table 5 Results of investigation on the linear relationship of compounds 2,5-DOF and 2,6-DOF

[0031]

[0032]

[0033] 2.4.2 Limit of detection and limit of quantification

[0034] Accurately pipette the No. 6 mixed standard solution prepared in "2.4.1", in which the concentrations of 2,5-DOF and 2,6-DOF are both 0.019 mg / mL, and dilute them step by step with 75% methanol solution and record the concentrations of the two compounds in each level of mixed standard solution. Pass the sample through a 0.22μm microporous filter membrane before injection, and then take 20μL of the filtrate and inject it into the HPLC chromatograph and record the signal-to-noise ratio (S / N). When the signal-to-noise ratio (S / N) is ≧3.0, the corresponding injection mass is the detection limit; when the signal-to-noise ratio (S / N) is ≧10.0, the corresponding injection mass is the quantification limit. The experimental results are shown in Table 6.

[0035] Table 6 Detection limit and quantification limit experimental results of compounds 2,5-DOF and 2,6-DOF

[0036]

[0037] 2.4.3 Precision

[0038] Select the mixed standard solution of 2,5-DOF and 2,6-DOF prepared in "2.4.1" with concentrations of 0.096 and 0.095 mg.mL-1, respectively, pass through a 0.22μm microporous filter membrane, and use a microinjection needle to accurately transfer 20μL of the filtrate and inject it into the HPLC chromatograph. Inject 6 times continuously on the same day, record the chromatographic peak areas of 2,5-DOF and 2,6-DOF, calculate the relative standard deviation (RSD), which is the intra-day precision, and the results are shown in Table 7. In the same way, inject 2 times on the same day, and inject for 3 consecutive days, record the chromatographic peak areas of 2,5-DOF and 2,6-DOF, calculate the relative standard deviation (RSD), which is the inter-day precision, and the results are shown in Table 8. The results show that the RSD values ​​of the chromatographic peak areas of 2,5-DOF and 2,6-DOF are less than 2.0%, indicating that the instrument has good precision.

[0039] Table 7 Intra-day precision results of compound 2,5-DOF and 2,6-DOF

[0040]

[0041]

[0042] Table 8 Results of daytime precision study of compound 2,5-DOF and 2,6-DOF

[0043]

[0044] 2.4.4 Repeatability

[0045] According to the sample solution preparation method in "2.3", 6 test sample solutions were prepared, filtered through a 0.22μm microporous filter membrane, and 20μL of the filtrate was accurately pipetted into the HPLC chromatograph using a microinjection needle, and the peak area was recorded. The content and RSD values ​​of the target compounds 2,5-DOF and 2,6-DOF were calculated by the external standard method. The results are shown in Table 9. The results show that the RSD values ​​are all less than 2.0%, indicating that the repeatability of this method is good.

[0046] Table 9 Repeatability test results of compounds 2,5-DOF and 2,6-DOF

[0047]

[0048] 2.4.5 Stability

[0049] According to the sample solution preparation method in "2.3", one sample solution was prepared, and the sample solution was filtered through a 0.22 μm microporous membrane. Every 2 hours, 20 μL of the filtrate was accurately transferred into the HPLC chromatograph using a microinjection needle. The measurement was performed 6 times in total, and the retention time (T) and peak area (S) of the target compounds 2,5-DOF and 2,6-DOF were recorded. The RSD values ​​were calculated respectively, and the results are shown in Table 10. The experimental results show that the RSD values ​​of the retention time and peak area of ​​2,5-DOF and 2,6-DOF are both less than 2.0%, indicating that the sample solution has good stability.

[0050] Table 10 Results of stability study of compounds 2,5-DOF and 2,6-DOF

[0051]

[0052] Table 10

[0053]

[0054] 2.4.6 Sample recovery rate

[0055] Weigh 6 portions of Hovenia dulcis, 2.5 g each (the contents of 2,5-DOF and 2,6-DOF in Hovenia dulcis are 0.048 mg and 0.053 mg, respectively), and randomly divide the 6 samples into three groups, 2 portions in each group. Weigh 80%, 100%, and 120% of the standard of 2,5-DOF and 2,6-DOF in Hovenia dulcis, and add them to the three groups of samples, respectively. Prepare 6 portions of spiked test solution according to the sample solution preparation method of "2.3", pass through a 0.22 μm microporous filter membrane, and accurately transfer 20 μL of the filtrate with a microinjection needle to the HPLC chromatograph, measure three times in parallel, record the peak areas of the target compounds 2,5-DOF and 2,6-DOF, and calculate the content, recovery rate, and RSD value of the target compounds 2,5-DOF and 2,6-DOF by the external standard method. The results are shown in Tables 11 and 12.

[0056]

[0057] Table 11 Results of sample recovery of compound 2,5-DOF

[0058]

[0059] Table 12 Results of sample recovery of compound 2,6-DOF

[0060]

[0061] The experimental results showed that the average recoveries of compounds 2,5-DOF and 2,6-DOF were 101.64% and 99.31%, respectively, and their RSD values ​​were 1.51% and 2.02%, respectively. Both RSD values ​​were less than 3%, indicating that the recovery rate of this method was good.

[0062] 2.5 Determination of 2,5-DOF and 2,6-DOF in Hovenia dulcis

[0063] 2.5.1 Chromatographic conditions

[0064] The chromatographic column was a Unitary NH2 (5μm, 100A, 4.6×250mm) amino column, the mobile phase was acetonitrile-water (89:11), the flow rate was 1.0mL / min; the column temperature was 30°C; the detection wavelength was 275nm.

[0065] 2.5.2 Preparation of standard solution

[0066] Accurately weigh 5.1 mg and 5.1 mg of the reference compounds 2,5-DOF and 2,6-DOF, respectively, add a small amount of 75% methanol solution to dissolve, transfer to a 100 mL volumetric flask, make up to volume with 75% methanol solution, mix well, and pass through a 0.22 μm microporous filter membrane to obtain mixed standard solutions with a concentration of 0.048 mg.mL-1 for 2,5-DOF and 0.048 mg.mL-1 for 2,6-DOF.

[0067] 2.5.3 Preparation of test solution

[0068] Weigh 5.0g of Hovenia dulcis A, B, and C (A: purchased from Bozhou, Anhui; B: purchased from Anguo, Hebei; C: purchased from Changchun, Jilin) ​​respectively, and prepare the test solution according to the sample solution preparation method in "2.3": heat and reflux with pure water for extraction 3 times, each time with a solid-liquid ratio of 10 times (W / V, g / mL), and the extraction time is 2h, 1h, and 1h. Combine the extracts, filter, and pass the filtrate through a 30.0g LK1300S SPE column, elute with 0.2% NaOH solution (m / V, g / mL), the eluent volume is 400mL, rinse with pure water until neutral, and then elute with 60% ethanol solution, the eluent volume is 260mL. Recover the solvent from the eluate until it is dry, dissolve it with a small amount of 75% methanol solution by ultrasonication, transfer it to a 2 mL volumetric flask, make up to volume with 75% methanol solution, shake well, and filter through a 0.22 μm microporous filter membrane to obtain the test solution.

[0069] 2.5.4 Content determination

[0070] According to the chromatographic conditions described in "2.5.1", 20 μL of the test solution and the standard solution were injected, and each sample was injected twice in parallel. The chromatographic peak area was recorded, and the content of the selected compound was calculated using the external standard one-point method. The results are shown in Table 13

[0071] Table 13 The contents of compounds 2,5-DOF and 2,6-DOF in different purchased Hovenia dulcis seeds

[0072] BRIEF DESCRIPTION OF THE DRAWINGS

[0073] Figure 1 . 1H-NMR spectrum of 2,5-DOF

[0074] Figure 2 . 13C-NMR spectrum of 2,5-DOF

[0075] Figure 3 . 2,5-DOF DEPT90°, DEPT135° maps

[0076] Figure 4 . 1H-NMR spectrum of 2,6-DOF

[0077] Figure 5 . 13C-NMR spectrum of 2,6-DOF

[0078] Figure 6 . 2,6-DOF DEPT90°, DEPT135° maps

[0079] Figure 7 . HPLC chromatogram of 2,5-DOF and 2,6-DOF standards

[0080] Figure 8 .HPLC chromatogram of Hovenia dulcis extract

[0081] Fig. 9 . 2,5-DOF structural formula

[0082] Fig.10 . 2,6-DOF structural formula DETAILED DESCRIPTION

[0083] The present invention will be further described below with reference to specific embodiments.

[0084] Example 1

[0085] 10 kg of Hovenia dulcis fruit was taken, crushed, and extracted by reflux with water for 3 times, with 100 L of water added each time. The filtrate was filtered, combined, and adsorbed on a D101 macroporous adsorption resin. The filtrate was first eluted with a 0.2% sodium hydroxide aqueous solution, rinsed with pure water until neutral, and then eluted with an 85% ethanol solution. The ethanol eluate was decompressed and the solvent was recovered to obtain an extract. The extract was subjected to a first silica gel column chromatography with a mixed solution of ethyl acetate-ethanol-water in a volume ratio of 60:2:1 to obtain a component (component A) containing 2,5-DOF and 2,6-DOF; component A was taken and subjected to a second silica gel column chromatography with a mixed solution of ethyl acetate-ethanol-water in a volume ratio of 5:4:0.4 as the mobile phase to obtain a component containing 2,5-DOF and 2,6-DOF. OF component (component B); component B was subjected to a third silica gel column chromatography with a mixed solution of dichloromethane-methanol-water in a volume ratio of 76:8:1 to obtain a component containing 2,5-DOF and 2,6-DOF (component C); component C was subjected to a fourth silica gel column chromatography with a mixed solution of dichloromethane-methanol-water in a volume ratio of 15:3:0.4 as the mobile phase to obtain a component containing 2,5-DOF and 2,6-DOF (component D); component D was separated and purified by preparative high performance liquid chromatography using an amino chromatographic column with acetonitrile-water (89:11, V / V) as the mobile phase to obtain 64.2 mg of 2,5-DOF monomer compound and 40.7 mg of 2,6-DOF monomer compound. The obtained monomer compounds were detected by HPLC-UV, and their purities were 95.4% and 93.3%, respectively.

[0086] Example 2

[0087] Take 10kg of Hovenia dulcis, crush it, add water and heat it under reflux for extraction 3 times, each time the solid-liquid ratio is 10 times (W / V, g / mL), and the extraction time is 2h, 1h, and 1h. Combine the extracts, filter, and adsorb the filtrate through 15kg of LK1300S macroporous adsorption resin, first elute with 0.2% NaOH solution (m / V, g / mL), rinse with pure water until neutral, and then elute with 60% methanol solution. The methanol eluent is decompressed and the solvent is recovered to obtain the extract. The extract is subjected to the first silica gel column chromatography with a mixed solution of ethyl acetate-ethanol-water in a volume ratio of 60:2:1 to obtain a component containing 2,5-DOF and 2,6-DOF (component A); component A is taken and subjected to the second silica gel column chromatography with a mixed solution of ethyl acetate-ethanol-water in a volume ratio of 5:4:0.4 as the mobile phase to obtain a component containing 2,5-DOF and 2,6-DOF ( Component B); Component B was subjected to a third silica gel column chromatography with a mixed solution of dichloromethane-methanol-water in a volume ratio of 69:8:1 to obtain a component containing 2,5-DOF and 2,6-DOF (component C); Component C was subjected to a fourth silica gel column chromatography with a mixed solution of dichloromethane-methanol-water in a volume ratio of 15:3:0.4 as the mobile phase to obtain a component containing 2,5-DOF and 2,6-DOF (component D); Component D was separated and purified by preparative high performance liquid chromatography using an amino chromatographic column with acetonitrile-water (89:11, V / V) as the mobile phase to obtain 56.7 mg of 2,5-DOF monomer compound and 27.9 mg of 2,6-DOF monomer compound. The obtained monomer compounds were detected by HPLC-UV, and their purities were 96.8% and 92.6%, respectively.

[0088] Example 3

[0089] 1 Chromatographic conditions

[0090] The chromatographic column was a Unitary NH2 (5μm, 100A, 4.6×250mm) amino column, the mobile phase was acetonitrile-water (89:11, V / V), the flow rate was 1.0mL / min; the column temperature was 30°C; the detection wavelength was 275nm.

[0091] 2. Preparation of standard solution

[0092] Accurately weigh 10.1 mg and 10.2 mg of standard samples of 2,5-DOF (purity of 95.4%) and 2,6-DOF (purity of 93.3%), respectively. Add a small amount of 75% methanol solution and sonicate until completely dissolved. Transfer the solution to a 10 mL volumetric flask, make up to volume with 75% methanol solution and shake well to obtain a mixed standard solution with concentrations of 0.964 mg.mL-1 and 0.952 mg.mL-1 for 2,5-DOF and 2,6-DOF, respectively.

[0093] 3 Preparation of test solution

[0094] Take 5g of Hovenia dulcis powder and reflux extract 3 times. The extraction solvent is pure water. The extraction time is 2h, 1h, and 1h respectively. The amount of pure water is 50mL. Combine the 3 extracts and filter them. Dilute with water, adsorb on a 30gLK1300S macroporous adsorption resin column, elute with 0.2% NaOH solution, rinse with water to neutrality, desorb with 60% ethanol, collect the eluate, concentrate to dryness under reduced pressure, dissolve with an appropriate amount of 75% methanol ultrasonically and make up to 2mL volumetric flask, shake well to obtain the test solution for later use.

[0095] 4. Content determination

[0096] According to the chromatographic conditions described in "1", the contents of compounds 2,5-DOF and 2,6-DOF in three samples of Hovenia dulcis purchased from different places (A: purchased from Bozhou, Anhui; B: purchased from Anguo, Hebei; C: purchased from Changchun, Jilin) ​​were determined. The contents of compound 2,5-DOF in Hovenia dulcis purchased from different places were as follows: Jilin > Hebei > Anhui, and their contents were 0.022 mg.g-1, 0.021 mg.g-1, and 0.008 mg.g-1, respectively, with an average content of 0.017 mg.g-1; while the contents of compound 2,6-DOF were as follows: Hebei > Jilin > Anhui, and their contents were 0.020 mg.g-1, 0.019 mg.g-1, and 0.015 mg.g-1, respectively, with an average content of 0.018 mg.g-1.

[0097] In summary, the present invention discloses a preparation method for separating and extracting compounds 2,5-DOF and 2,6-DOF from Hovenia dulcis, and a new SPE-HPLC-UV determination method for determining the content of 2,5-DOF and 2,6-DOF in Hovenia dulcis. It should be clarified here that for those skilled in the art, with reference to the research process and embodiments disclosed in the present invention, they can still improve and modify the technical solutions described in the present invention and the aforementioned embodiments, or make equivalent substitutions for some of the technical features therein. It should be emphasized here that any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for preparing 2,5-deoxyfructozazine (2,5-DOF) and 2,6-deoxyfructozazine (2,6-DOF), characterized in that The raw material used to prepare 2,5-DOF and 2,6-DOF is Hovenia dulcis.

2. The preparation method according to claim 1, characterized in that The extraction is performed with water or an aqueous solution of an organic solvent, the aqueous extract is directly adsorbed by a macroporous adsorption resin, the organic solvent extract is decompressed to recover the solvent, diluted with water, and then adsorbed by a macroporous adsorption resin, after the adsorption is completed, it is first eluted with alkaline water and then eluted with an alcohol solution, the alcohol eluent is decompressed to recover the solvent to obtain an extract, and the extract is separated by normal phase column chromatography.

3. The preparation method according to claim 2, characterized in that The macroporous adsorption resin is one of HP-20, D101 or LK1300S macroporous adsorption resins or a mixture of two or more thereof; the alcohol solution is ethanol or methanol solution; and the normal phase column chromatography stationary phase is silica gel or amino-bonded fully porous spherical ultrapure silica gel.

4. The preparation method according to claim 2, characterized in that The mobile phase of the silica gel column chromatography is a mixed solution of ethyl acetate-ethanol-water with a volume ratio of 60:2:1 to 5:4:0.4 or a mixed solution of dichloromethane-methanol-water with a volume ratio of 76:8:1 to 15:3:0.

4.

5. The method according to claim 2, characterized in that The separation and purification of the amino-bonded fully porous spherical ultrapure silica gel was carried out by preparative high performance liquid chromatography, and the mobile phase was acetonitrile-water (89:11, V / V).

6. A method for determining the content of 2,5-DOF and 2,6-DOF in Hovenia dulcis by solid phase extraction-high performance liquid chromatography-ultraviolet detector (SPE-HPLC-UV), characterized in that The SPE stationary phase is LK1300S macroporous adsorption resin, the chromatographic column is an amino chromatographic column, and the mobile phase is an acetonitrile-water mixed solution.

7. The method for determining the content of the compound according to claim 6, characterized in that The mobile phase used was acetonitrile-water (89:11, V / V), the flow rate was 1.0 mL / min, and the column temperature was 30°C.

8. The method for determining the content of the compound according to claim 6, characterized in that The preparation method of the test solution is as follows: take 5 g of Hovenia dulcis powder, extract it with pure water reflux for 3 times, the extraction time is 2h, 1h, and 1h respectively, the amount of pure water is 50mL, the 3 extracts are combined and filtered, the filtrate is adsorbed on a LK1300S macroporous adsorption resin column, rinsed with 0.2% NaOH solution to remove flavonoids and other pigment impurities, desorbed with 60% ethanol, the eluate was collected, concentrated to dryness under reduced pressure, dissolved in 75% methanol and diluted to a 2mL volumetric flask, and shaken to obtain.

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