A preparation method of 5-(glucosyl-α-1-6-glucose)-hydroxymethylfurfural

Abstract

The invention discloses a method for preparing 5-(glucose-α-1-6-glucose)-hydroxymethylfurfural. The method comprises the following steps: heating sugar to make it smolder, passing the smoke generated by the smoldering into pure water or a low-concentration ethanol solution, desorbing after adsorption with a macroporous adsorption resin, and further purifying using a semi-preparative HPLC method or a silica gel column chromatography method; the purified solution is rotary evaporated and freeze-dried to obtain 5-GGMF. The present invention replaces Radix Rehmanniae Preparata with common food-grade sugar raw materials, and synthesizes the drug component through heating, recycling purification, rotary evaporation, and freeze drying in four steps, which has the characteristics of easy availability of raw materials, simple steps, high yield, low production cost, and less three wastes, and has a good prospect for industrial application.

Description

Technical Field

[0001] The invention relates to a method for preparing 5-(glucosyl-α-1-6-glucose)-hydroxymethylfurfural. Background Art

[0002] As one of the "Four Major Huai Medicines", Rehmannia is a widely used traditional Chinese medicine. According to whether it is processed or not, the Chinese Pharmacopoeia divides Rehmannia into fresh Rehmannia, raw Rehmannia and cooked Rehmannia. Raw Rehmannia is fresh Rehmannia that has been baked to about 80% dry, and it mainly has the effects of stopping bleeding and lowering blood sugar. If raw Rehmannia is further heated (using auxiliary materials such as rice wine, tangerine peel, and Amomum villosum, and mainly processed by steaming, boiling, and stewing), it will be processed into cooked Rehmannia, which has a warm nature and its effect changes from clearing to nourishing, mainly to nourishing blood. Wu Jinhuan et al. (2011) found that raw Rehmannia had a more significant blood sugar lowering effect and blood lipid improvement level on diabetic mice induced by streptozotocin than cooked Rehmannia. Pu-Yang Gong et al. (2019) found that raw Rehmannia can anti-coagulate blood and inhibit thrombosis, while cooked Rehmannia has a significant blood nourishing effect and can effectively improve the peripheral blood count of chronic aplastic anemia. Zhou Li et al. (2016) found that during the processing of Rehmannia glutinosa, the content of most functional ingredients decreased significantly, and only the content of furan aldehyde derivatives such as 5-hydroxymethylfurfural (5-HMF) and its glycosylated derivatives (5-glucosylhydroxymethylfurfural, 5-GMF and 5-(glucosyl-α-1-6-glucose)-hydroxymethylfurfural, 5-GGMF) increased significantly. Glycosylated derivatives of furan aldehydes are the main components of Rehmannia glutinosa with anemia treatment effects. Song-Lin Li et al. (2010) tested the content of a wide variety of medicinal ingredients in Rehmannia glutinosa and found that leonurin and its isomers are the characteristic medicinal ingredients of raw Rehmannia glutinosa, while 5-GGMF is the characteristic medicinal ingredient of cooked Rehmannia glutinosa. Before this, the content of verbascoside and 5-HMF was mainly used as the evaluation criteria for raw and cooked Rehmannia glutinosa. In fact, 5-HMF is a common component in many processed herbal medicines, so it is no longer recommended to use it as the characteristic medicinal ingredient of cooked Rehmannia glutinosa.

[0003] Since 5-GGMF is the signature drug component of Rehmannia glutinosa, we have studied a more economical and effective way to obtain it. At present, there is no chemical synthesis method for 5-GGMF, and it can only be extracted from Rehmannia glutinosa. The content of this drug component in Rehmannia glutinosa generally does not exceed 0.3%. Tae Hyung Won et al. (2014) used HPLC-DAD-ESI-MS to quantitatively analyze furanaldehyde derivatives in 18 types of Rehmannia glutinosa commercially available in China and South Korea, and measured the highest 5-GGMF content of 2345 μg / g (0.23%) and the lowest 5-GGMF content of 196 μg / g (0.02%). The wholesale price of raw Rehmannia glutinosa in the medicinal material market is generally 6 to 10 yuan / jin, and Rehmannia glutinosa is about 16 to 25 yuan / jin. Therefore, if Rehmannia glutinosa is used as a raw material to extract 5-GGMF, the preparation process is not only complicated, time-consuming and labor-intensive, but also too costly. Summary of the invention

[0004] In view of the above-mentioned technical problems, the technical solution of the present application heats sugars at a certain temperature and allows them to smolder, and the smoke produced contains a large amount of 5-GGMF. The component can be prepared by recovering, extracting and purifying it by a suitable method. The yield of 5-GGMF is about 3% based on sucrose as the raw material.

[0005] The purpose of the present invention can be achieved through the following technical solutions:

[0006] A method for preparing 5-(glucosyl-α-1-6-glucose)-hydroxymethylfurfural, the method comprising the following steps: heating sugar to make it smolder, passing smoke generated by the smoldering into pure water or ethanol solution, then adsorbing with a macroporous adsorption resin and then desorbing, and purifying with semi-preparative HPLC or silica gel column chromatography; rotary evaporating the purified solution and freeze-drying it to obtain 5-GGMF.

[0007] In the technical solution of the present invention: the sugar is at least one of monosaccharide, disaccharide and trisaccharide. Further: the sugar is sucrose.

[0008] In the technical solution of the present invention: the temperature at which the sugar is heated to smolder is 200°C to 600°C. Further: the temperature at which the sugar is heated to smolder is 300°C to 350°C.

[0009] In the technical solution of the present invention: the concentration of the ethanol solution is 0.01-20%.

[0010] In the technical solution of the present invention: the type of the macroporous adsorption resin is X-5 type, AB-8 type, XAD-7 type, DA-201 type, D4020 type, D101 type or NKA-9 type; preferably, the type of the macroporous adsorption resin is XAD-7 type.

[0011] In some specific technical solutions: ethanol with a volume concentration of 60% to 90% is used for desorption, and the desorption temperature is 30 to 50°C.

[0012] In the technical solution of the present invention: the temperature of the rotary evaporation is 55-75°C; preferably, the temperature of the rotary evaporation is 65°C.

[0013] In the technical solution of the present invention, the mass volume ratio of sugar to pure water or ethanol solution is 1g:10-20mL.

[0014] Beneficial effects of the present invention:

[0015] The present application uses common food-grade sugars such as sucrose instead of Rehmannia glutinosa as raw materials, which have a wide source and low preparation cost;

[0016] In the method of the present invention, macroporous adsorption resin, semi-preparative HPLC method or silica gel column chromatography can be used for large-scale continuous separation and purification, and 5-GGMF with sufficient purity can be obtained, and the purification cost is low;

[0017] The preparation process of the invention is simple, less three wastes and low energy consumption. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The figure is a schematic diagram of the process flow of 5-GGMF preparation and purification of the present invention;

[0019] Figure 2 This is the ultraviolet spectrum analysis chart of 5-GGMF prepared in Example 1 of the present invention;

[0020] Figure 3 This is the mass spectrum of 5-GGMF prepared in Example 1 of the present invention;

[0021] Figure 4 The monosaccharide composition analysis diagram of 5-GGMF prepared in Example 1 of the present invention: (a) chromatographic elution curve of standard glucose; (b) chromatographic elution curve of 5-GGMF;

[0022] Figure 5 This is a near-infrared spectrum of 5-GGMF prepared in Example 1 of the present invention;

[0023] Figure 6 The nuclear magnetic resonance spectra of 5-GGMF prepared in Example 1 of the present invention are as follows: (a) hydrogen nuclear magnetic resonance spectrum; (b) carbon nuclear magnetic resonance spectrum;

[0024] Figure 7 This is a schematic diagram of the molecular structure of 5-GGMF prepared in Example 1 of the present invention. DETAILED DESCRIPTION

[0025] The present invention is further described below in conjunction with embodiments, but the protection scope of the present invention is not limited thereto:

[0026] Embodiment 1:

[0027] Weigh 50g of sucrose, heat it to 330℃, pass the smoke generated by heating into 600mL of aqueous solution, adsorb it with 200g of XAD-7 macroporous adsorption resin, and then desorb it with 85% volume concentration ethanol solution at 40℃. Then purify it with semi-preparative HPLC, rotary evaporate the purified solution at 65℃, and freeze-dry it to obtain a reddish brown solid powder.

[0028] like Figures 2 to 7 The molecular structure of the product was analyzed using ultraviolet-visible spectroscopy, elemental analysis, high performance liquid chromatography, ultra-high performance liquid chromatography-mass spectrometry, Fourier transform near-infrared spectroscopy, and nuclear magnetic resonance spectroscopy. After analysis, the molecule was identified as 5-GGMF.

[0029] Figure 2 The ultraviolet spectrum of the product obtained by the present invention. The product has only one typical characteristic absorption peak at 280nm in the wavelength range of 200-600nm, which indicates that it has a strong chromophore in the ultraviolet region. According to the typical absorbance and intensity of the chromophore of organic compounds, the energy levels of the large π bonds of organic compounds with conjugated double bonds are close to each other, and the electrons are easily excited, which can produce an obvious K absorption band in the range of 210-280nm. In addition, for organic compounds containing ketones, the electronic transition is usually n-π*, which may also produce a weak absorption band at 280nm. Therefore, it can be preliminarily inferred that the structural system of the product may contain a conjugated double bond system or a ketone structure. On the other hand, for the final product of the caramelization reaction, Mendoza and his colleagues found that caramel pigments have specific absorption peaks at 280nm and 560nm after analyzing the physical and chemical properties of different types of commercially available caramels (caramel pigments Ⅰ, Ⅱ, Ⅲ and Ⅳ), among which 280nm is its main absorption peak. It is worth noting that the product has only a specific absorption peak around 280nm, which indicates that the product is most likely the end product of the caramelization reaction. Therefore, through UV spectrum analysis, it can be preliminarily inferred that the product may be the end product of the caramelization reaction and an unsaturated compound with a ketone or conjugated double bond structure system.

[0030] Table 1 is the elemental analysis results of 5-GGMF prepared in Example 1 of the present invention

[0031]

[0032] Table 1 shows the elemental analysis results of the product obtained by the present invention. The actual measured element content is not much different from the theoretical element content under the condition of 100% purity. According to calculation, the purity of the product is above 99.42% by using this set of separation and purification procedures.

[0033] Figure 3 The mass spectrum result of the product obtained by the present invention. It can be found that the product obtains the highest quasi-molecular ion peak intensity at 449.00. In addition, neutral losses 357.98, 241.02, and 164.03 are also found in the fragment ion peak. According to the molecular ion peak m / z 449 in the mass spectrum, the molecular weight of the product can be determined to be 449, which further indicates that the product may be a volatile caramel colorant rather than a volatile flavor compound from high temperature. For example, furfural (molecular weight: 96.08), 5-hydroxymethylfurfural (molecular weight: 126.11), etc. In recent years, some scholars have attempted to construct a caramel formation hypothesis by artificially searching for caramelization products of glucose, fructose and sucrose. For example, Agnieszka Golon et al. analyzed the products of caramelization reactions of glucose, fructose and sucrose by combining high-resolution mass spectrometry with Van Krevelen analysis, Kendrick analysis and homologous sequence analysis. It was found that the products of the caramelization reaction of glucose, fructose and sucrose mainly include the dehydration products of monomeric hexose, the dehydration products of oligomeric hexose, the hydration products of oligomeric hexose, the fragment products produced by the redox disproportionation reaction and a small amount of aromatic compounds. At the same time, for the dehydration products of monomeric hexose and oligomeric hexose, the mass spectral information corresponding to the loss of one molecule of water (m / z 161.1) and the loss of three molecules of water (m / z 287.1, 449.2, 611.2, 773.3 and 935.3) in the pseudomolecular ion is the most abundant and the MS intensity is the highest. It is worth noting that according to the mass spectral information of the product, it can be found that the mass-to-charge ratio of 449.00 is very likely to be the product of the removal of three molecules of water from trimeric hexose (thermal decomposition products of glucose, fructose or sucrose). At the same time, the neutral loss of 164.03 is also likely to be caused by the loss of one molecule of water in the monomeric hexose. Combined with the previous UV-visible spectrum analysis, it is speculated that the product is most likely a dehydration polymerization product of glucose, fructose or sucrose, and its chemical formula is determined to be C 18 H 26 O 13 .

[0034] Figure 4 The HPLC elution curve shows that the product contains monosaccharide structures. By comparing the retention time of the product sample with the retention time of the standard monosaccharide, it is found that the retention time of the monosaccharide structure in the product (9.37 minutes) is the same as that of the standard glucose ( Figure 4-3), the results showed that the main monosaccharide structure in the product was glucose. It is generally believed that sucrose is usually hydrolyzed into glucose and fructose by cleaving glycosidic bonds, and then glucose and fructose are further dehydrated in other forms to form caramel. According to the monosaccharide composition analysis, the main monosaccharide composition of the product is glucose rather than fructose.

[0035] Figure 5 The Fourier transform infrared spectrum of the product obtained in the present invention is shown in Fig. -1 FTIR absorption spectrum of the product. First, it can be observed from 3600cm -1 To 3200cm -1 There is a very broad band, which is mainly caused by OH stretching vibration. In addition, it can be found from the spectrum that the total intensity of OH stretching vibration dominates all other bands, indicating that the content of hydroxyl moieties in the product is high. This may be caused by the large amount of hydroxyl stretching vibration in glucose. Interestingly, the 3080cm -1 NH + This further indicates that the product does not contain protein or Maillard reaction products. On the other hand, at 1739 cm -1 The absorption peak at 1604cm -1 The strong stretching at is attributed to the C=C double bond stretching. These results indicate that there may be furan structures in the product. Because, previous studies have shown that rapid pyrolysis of glucose, fructose, and sucrose can produce various furan compounds, among which 5-hydroxymethylfurfural and furfural are the two most important products. In addition, it is worth noting that both 5-hydroxymethylfurfural and furfural contain aldehyde groups and conjugated double bonds, and the aldehyde groups and conjugated double bonds in 5-hydroxymethylfurfural and furfural are formed by removing three molecules of water from hexose. At the same time, according to the mass-to-charge ratio of the previous product, the C 18 H 26 O 13 The sum of the formula is equal to three molecules of glucose minus three molecules of water. Therefore, it can be inferred that the molecular structure of the product may contain the structural system of furfural or 5-hydroxymethylfurfural. -1 and 2852cm -1 The absorption peaks at 1050cm represent the stretching vibrations of CH2 and CH. -1 The absorption peak at 862 cm -1 The absorption peak at is an α-glycoside band, indicating that the product may contain an α-pyranose ring.

[0036] Figure 6 The nuclear magnetic resonance spectrogram of the product obtained by the present invention. As can be seen from the figure, the product has obvious chemical shifts in the nuclear magnetic resonance spectrum. The chemical shifts at 182.27 and 9.21ppm in the nuclear magnetic resonance spectrum belong to the carbon atoms and hydrogen atoms in the aldehyde group of 5-hydroxymethylfurfural. In addition, the signals at 126.76, 110.86, 7.30 and 6.45ppm are carbon atoms and hydrogen atoms in the conjugated double bonds in the furan structure. This research result is consistent with the results of the structural analysis of the product in the UV-Vis spectrum and Fourier transform spectrum. On the other hand, the signals at 161.23 and 151.64ppm may belong to two carbon atoms of COC. 13 The main signal peak at 101.28 ppm in the C spectrum indicates the presence of the α-anomeric carbon in α-pyranose glucose in the product. At the same time, this end-isomerization signal indicates that the product is connected by an α-1-6-glycosidic bond. In addition, some studies have also found that the signal peak at 61.00-85.60 ppm may be attributed to the C-2 to C-6 part of the glucose residue. 13 The signal peaks of C NMR spectrum at 66.09, 69.96, 70.60, 72.32, and 76.12 ppm respectively indicate that C-4, C-6, C-2, C-5, and C-3 of the glucose residues in the product are substituted. 1 Other signal peaks in the H NMR spectrum, the triplet at 5.24 ppm, are likely to be derived from the hydrogen atoms of the glucose hydroxyl groups. On the other hand, the obvious hydrogen proton signal at 4.46 ppm is attributed to the methylene group in the side chain of 5-hydroxymethylfurfural. 1 The hydrogen proton signal (5.56ppm) present in the terminal hydroxyl structure of 5-hydroxymethylfurfural was not observed in the HNMR spectrum. The possible reason for this phenomenon is that the terminal hydroxyl group of 5-hydroxymethylfurfural and the hemiacetal hydroxyl group at the C1 position of glucose are converted into α-1-6 glycosidic bond-linked glucosyl hydroxymethylfurfural through intermolecular dehydration. In addition, 1 The other signal peaks of the H NMR spectrum at 3.90, 3.88, 3.79, and 3.62 ppm respectively represent substitutions at H-5, H-2, H-6, and H-4 in the glucose residue.

[0037] The yield of 5-GGMF was 3.04% based on sucrose as raw material, and the purity of 5-GGMF was 99.42% based on elemental analysis results.

[0038]

[0039] Embodiment 2:

[0040] Weigh 50g of sucrose, heat to 330℃ with ultrasound assistance, pass the smoke generated by heating into 600mL of aqueous solution, purify with 200g of XAD-7 macroporous adsorption resin, and then desorb with 85% ethanol solution by volume, and the desorption temperature is 40℃. Then purify with semi-preparative HPLC, rotary evaporate the purified solution at 65℃, and freeze-dry to obtain a reddish brown solid powder. The yield of 5-GGMF calculated with sucrose as raw material is 3.27%, and the purity is 97.06%.

[0041] Embodiment 3:

[0042] Weigh 50g of sucrose, heat it to 330℃, pass the smoke generated by heating into 600mL of aqueous solution, purify it with 200g of XAD-7 macroporous adsorption resin, and then desorb it with ethanol solution with a volume concentration of 85%, and the desorption temperature is 40℃. Then purify it with silica gel chromatography (Kiesel gel#60, 70-230mesh). The purified solution is rotary evaporated at 65℃ and freeze-dried to obtain a reddish brown solid powder. The yield of 5-GGMF calculated with sucrose as raw material is 2.65%, and the purity is 99.53%.

[0043] The above is only a preferred embodiment of the present invention. It should be pointed out that a person skilled in the art can make several modifications and improvements without departing from the inventive concept of the present invention, which all belong to the protection scope of the present invention.

Claims

1. A method for preparing 5-(glucosyl-α-1-6-glucose)-hydroxymethylfurfural, characterized in that: The method comprises the following steps: heating sugar to make it smolder, passing smoke generated by the smoldering into pure water or ethanol solution, then adsorbing with macroporous adsorption resin and then desorbing, purifying with semi-preparative HPLC or silica gel column chromatography; rotary evaporating and freeze-drying the purified solution to obtain 5-GGMF; The temperature at which the sugar is heated to smoldering is 200°C to 600°C; the type of the macroporous adsorption resin is X-5, AB-8, XAD-7, DA-201, D4020, D101 or NKA-9; ethanol with a volume concentration of 60% to 90% is used for desorption, and the desorption temperature is 30 to 50°C; the temperature of the rotary evaporation is 55 to 75°C.

2. The method according to claim 1, characterized in that: The sugar is at least one of monosaccharide, disaccharide and trisaccharide.

3. The method according to claim 2, characterized in that: The sugar is sucrose.

4. The method according to claim 1, characterized in that: The temperature at which sugars are heated to smolder is 300°C to 350°C.

5. The method according to claim 1, characterized in that: The concentration of the ethanol solution is 0.01-20%.

6. The method according to claim 1, characterized in that: The macroporous adsorption resin is of XAD-7 type.

7. The method according to claim 1, characterized in that: The temperature of the rotary evaporation was 65°C.

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