Application of Nano Zero-Valent Iron Catalyst in the Conversion of Rare Ginsenosides

Abstract

The present invention provides an application of a nano zero-valent iron catalyst in the conversion of rare ginsenosides. The nano zero-valent iron catalyst is prepared by the following steps: S1, taking dried Sophora japonica powder and adding a certain proportion of ethanol solution to a reaction vessel, heating and refluxing for extraction for a certain period of time, and then filtering the mixed solution to obtain a Sophora japonica extract; S2, taking the Sophora japonica extract prepared above, adding a certain mass of ferric chloride hexahydrate to a container, and performing hydrothermal synthesis for a certain period of time, centrifugal washing, and vacuum drying to obtain nano zero-valent iron particles; the nano zero-valent iron catalyst is used to convert ginsenoside Rb1 into rare ginsenoside derivatives 25-OH-Rg3. The advantages of the present invention are: the synthesis method of preparing a nano zero-valent iron catalyst by reducing trivalent iron ions with Sophora japonica extract is simple in process, low in energy consumption, and has a wide range of raw material sources. It has high specificity when used to catalyze the conversion of ginsenoside Rb1 into rare ginsenoside Rg3 derivatives, can be efficiently and quickly catalyzed, and has a high conversion rate.

Description

Technical Field

[0001] The invention belongs to the field of catalysts, and in particular relates to the application of a nano zero-valent iron catalyst in the transformation of rare ginsenosides. Background Art

[0002] Ginseng( Panax ginseng Ginseng (CA Meyer) is a plant of the genus Panax in the Araliaceae family. The medicinal ingredients are concentrated in the roots or rhizomes of ginseng. Ginseng saponins, amino acids, ginseng polysaccharides, organic acids and volatile oils are the main material components of ginseng. Among them, ginsenosides are the main active ingredients of ginseng, which have anti-tumor, antioxidant and central nervous system regulation effects. Rare ginsenosides have the characteristics of small relative molecular mass and high biological activity, showing better pharmacological activity, but the content of rare ginsenosides in ginseng is very low and can only be obtained by converting the main ginsenosides. The difference between the main ginsenosides and the rare ginsenosides is mainly reflected in the different glycosyl substituents connected to the sapogenin. The glycosyl of ginsenosides is closely related to their biological activity, and the reduction of the number of glycosylation may significantly improve the bioavailability of ginsenosides.

[0003] At present, the main methods for transforming rare ginsenosides are chemical transformation and biological transformation. Chemical transformation usually uses inorganic or organic acids and bases as catalysts to produce rare saponins by hydrolyzing and deglycosylation of ginsenosides in an acidic or alkaline environment; biological transformation usually uses biocatalysts such as glycosidases and microorganisms to hydrolyze glycosidic bonds to prepare rare saponins. Acid and alkaline hydrolysis and transformation reactions are violent, lack directionality and purposefulness, produce complex products, and pollute the environment. Biological transformation reactions are mild, produce single products, and are pollution-free, but the biological enzymes used require a lot of time for preliminary processes such as cultivation, extraction, and separation, and the storage time of the enzyme is short.

[0004] Yuan Ting et al. in the Journal of Dalian University of Technology "Metal Ion Catalytic Conversion of Ginsenoside Rg1 to Rh1" experimented with the effects of different metal ions and solvent systems on the conversion of ginsenoside Rg1, and mentioned that Fe 3+ Catalyze the conversion of ginsenoside Rb1 into rare ginsenoside Rg3 under mild reaction conditions, with relatively single products and no conversion rate mentioned; 3+ The catalytic reaction of ginsenoside Rg1 to produce rare ginsenoside Rh1 has stronger reaction specificity, more thorough substrate conversion and higher product yield. However, under the influence of different solvent systems, the conversion rate of ginsenoside Rg1 to produce rare ginsenoside Rh1 varies greatly. In a 50% ethylene glycol-water system, the yield of crude 20(S,R)-Rh1 is as high as 71.6% after 16h of catalytic reaction. When the catalysis is continued to 23h, the mass fraction of rare ginsenoside derivative 25(S,R)-OH-Rh1 in the product can reach the highest, about 40%.

[0005] The above methods still have room for improvement in the conversion efficiency of rare ginsenosides, especially rare ginsenoside derivatives, and the economy of the catalyst; therefore, it is very economical to provide a rare ginsenoside catalyst and catalytic conditions with high specificity, high conversion efficiency, short conversion time and no pollution. Summary of the invention

[0006] In view of the problems in the prior art of low specificity, long conversion time and low conversion rate of catalytic conversion of ginsenosides by rare ginsenoside catalysts, the present invention provides an application of a nano zero-valent iron catalyst in the conversion of rare ginsenosides.

[0007] The technical solution of the present invention is as follows: an application of a nano zero-valent iron catalyst in the transformation of rare ginsenosides, wherein the nano zero-valent iron catalyst is prepared by the following steps:

[0008] S1, taking dried Sophora japonica powder and adding a certain proportion of ethanol solution into a reaction vessel, heating and refluxing for extraction for a certain period of time, and then filtering the mixed solution to obtain Sophora japonica extract;

[0009] S2, adding a certain amount of ferric chloride hexahydrate to the prepared Sophora japonica extract in a container, performing hydrothermal synthesis for a certain period of time, performing centrifugal washing, and performing vacuum drying to obtain nano zero-valent iron particles;

[0010] The nano zero-valent iron catalyst is used to convert ginsenoside Rb1 into a rare ginsenoside derivative 25-OH-Rg3.

[0011] Furthermore, the liquid-to-solid ratio of the dry Sophora japonica flower powder to the ethanol solution is 10:1-50:1; and the concentration of the ethanol solution is 40%-100%.

[0012] Furthermore, the heating temperature is 50-90° C., and the reflux extraction time is 1-3 h.

[0013] Furthermore, the volume ratio of the mass of ferric chloride hexahydrate to the Sophora japonica flower extract is 2.7 mg / mL-54.0 mg / mL.

[0014] Preferably, the liquid-to-solid ratio of dried Sophora japonica buds to the extraction solvent is 20:1, and the concentration of the ethanol solution is 70%.

[0015] Preferably, the heating temperature is 80° C. and the reflux extraction time is 1 h.

[0016] Preferably, the mass ratio of ferric chloride hexahydrate to the Sophora japonica flower extract is 2.7 mg / mL.

[0017] Further, the application method includes:

[0018] Take the prepared nano zero-valent iron particles and add them to the ginsenoside Rb1 solution, and heat and convert them in a water bath shaker; after the reaction is completed, the reaction solution is placed at room temperature and filtered through a filter membrane to obtain a 25-OH-Rg3 derivative; the mass ratio of nano zero-valent iron particles to ginsenoside Rb1 is 1:0.02-0.08; the concentration of ginsenoside Rb1 solution is 10~100ppm; the heating temperature of the water bath shaker is 20~80℃, and the conversion time is 5~180min.

[0019] Preferably, the concentration of ginsenoside Rb1 solution is 40 ppm and the temperature of the water bath shaker is 80°C.

[0020] Preferably, the conversion time is 120 min.

[0021] The advantages of the present invention are: the synthesis method of preparing nano zero-valent iron catalyst by reducing trivalent iron ions with Sophora japonica extract is simple in process, low in energy consumption, and has a wide range of raw material sources. When used to catalyze the conversion of ginsenoside Rb1 into rare ginsenoside Rg3 derivatives, the specificity is high, and the efficient conversion of ginsenoside Rb1 can be completed in only 120 minutes, and the selectivity of rare ginsenoside Rg3 derivatives in the conversion products is very high. It provides an efficient and low-cost production route for preparing rare ginsenoside Rg3 derivatives. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 is the XRD spectrum of nano zero-valent iron particles;

[0023] Figure 2 This is the SEM&TEM spectrum of nano zero-valent iron particles;

[0024] Figure 3 This is the total ion flow diagram of ginsenoside Rb1 after 30 minutes of conversion under the catalysis of nano zero-valent iron particles extracted from Sophora japonica;

[0025] Figure 4 This is the secondary tandem spectrum of compound 1 in the total ion flow chart after 30 minutes of conversion of ginsenoside Rb1;

[0026] Figure 5 This is a comparison of the catalytic activity of nano zero-valent iron particles converted from different plant extracts on ginsenoside Rb1;

[0027] Figure 6 This is a comparison chart of serum uric acid levels in different groups of mice;

[0028] Figure 7 This is a comparison chart of serum creatinine levels in different groups of mice;

[0029] Figure 8 This is a comparison chart of serum urea nitrogen levels in different groups of mice;

[0030] Fig. 9 This is a comparison chart of xanthine oxidase (XOD) activity in different groups of mice;

[0031] Fig.10 This is a comparison chart of uric acid transporter levels in different groups of mice. DETAILED DESCRIPTION

[0032] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0033] Through in-depth research, the present invention found that when nano zero-valent iron prepared from Sophora japonica extract is used as a catalyst in the catalytic reaction process of rare ginsenosides, it has very high specificity and can efficiently and quickly catalyze the conversion of ginsenoside Rb1 into rare ginsenoside Rg3 derivatives.

[0034] Nano-Zero-Valent Iron (nZVI) refers to iron nanoparticles with a particle size of about 1-100nm. Compared with zero-valent iron, it has the characteristics of large specific surface area, strong surface activity, strong reduction ability and strong adsorption ability. It is currently used in pollution control.

[0035] The green synthesis method of reducing trivalent iron ions with green plant extracts to prepare nano zero-valent iron has been widely reported in recent years. Compared with the traditional synthesis methods of nano zero-valent iron (physical method, chemical method, etc.), it has the advantages of low energy consumption, simple process, and wide source of raw materials. Since there are many types of green plants that can prepare nano zero-valent iron, the advantages of nano zero-valent iron prepared by different green plants are also different.

[0036] Among them, Sophora japonica is the dried flower and bud of Sophora japonica, a leguminous plant, and has a wide range of sources. The main chemical components in Sophora japonica are flavonoids, saponins and fatty acid compounds. In addition, Sophora japonica also has some other chemical components, such as polysaccharides, volatile components, proteins, amino acids and trace elements.

[0037] The preparation method of the nano zero-valent iron catalyst provided by the present invention comprises the following steps:

[0038] S1, taking dried Sophora japonica powder and adding a certain proportion of extraction solvent into a reaction vessel, heating and refluxing for extraction for a certain period of time, and then filtering the mixed solution to obtain Sophora japonica extract;

[0039] S2. Add a certain amount of ferric chloride hexahydrate to the prepared Sophora japonica extract and place it in a container. After hydrothermal synthesis for a certain period of time, centrifugal washing is performed, and finally vacuum drying is performed overnight to obtain nano zero-valent iron particles.

[0040] The liquid-to-solid ratio of the dried Sophora japonica bud powder to the extraction solvent is 10:1 to 50:1. Preferably, the liquid-to-solid ratio of the dried Sophora japonica bud powder to the extraction solvent is 20:1.

[0041] The extraction solvent is an ethanol solution with a concentration of 40% to 100%, preferably, the concentration of the ethanol solution is 70%.

[0042] The heating temperature is 50-90° C., and the reflux extraction time is 1-3 h. Preferably, the heating temperature is 80° C., and the reflux extraction time is 1 h.

[0043] The mass ratio of ferric chloride hexahydrate to the sophora japonica extract is 2.7 mg / mL-54.0 mg / mL. Preferably, the mass ratio of ferric chloride hexahydrate to the sophora japonica extract is 27.0 mg / mL.

[0044] Figure 1 This is the XRD spectrum of nano-zero-valent iron particles (nZVI) prepared according to the above method. The diffraction peak mainly corresponds to FeCl2·4H2O (PDF # 74-0833), indicating that the generated nano-zero-valent iron exhibits strong reducing properties, reducing the added ferric chloride to ferrous chloride. This confirms the formation of nano-zero-valent iron and proves the reducing properties of organic compounds in Sophora japonica.

[0045] Figure 2 SEM & TEM spectra of nZVI prepared according to the above method. Figure 2 a and 2b are SEM images of nZVI. It can be seen that the nZVI synthesized using Sophora japonica extract shows a fine spherical or ellipsoidal morphology with a particle size distribution range of about 30-60 nm. Figure 2 c and 2d are TEM images of nZVI. The microstructure of the synthesized nZVI is clearly visible, and the black circular spots represent the synthesized nano-zero-valent iron.

[0046] The obtained nano zero-valent iron particles are used in the transformation of ginsenoside Rb1, and the method of use is as follows:

[0047] The prepared nano zero-valent iron particles are added to the ginsenoside Rb1 solution, and heated and converted in a water bath shaker; after the reaction is completed, the reaction solution is placed at room temperature and filtered through a filter membrane to obtain a mixture.

[0048] The concentration of ginsenoside Rb1 solution is 10~100ppm, preferably 40ppm; the mass ratio of nano zero-valent iron particles to ginsenoside Rb1 is 1:0.02-0.08; the water bath shaking temperature is 20~80℃, the conversion time is 5~180min, and the water bath shaking temperature is preferably 80℃; the pore size of the filter membrane is any one of 0.22μm, 0.45μm, and 0.60μm.

[0049] The obtained mixture was subjected to high performance liquid chromatography-mass spectrometry (HPLC-MS) analysis to determine its structure. The operation steps of high performance liquid chromatography-LTQ mass spectrometry are as follows:

[0050] In the HPLC procedure, the mobile phases were acetonitrile as phase A and 0.1% formic acid aqueous solution as phase B, both of which were chromatographically pure; the gradient elution conditions (volume fraction) were: 0~5 min (25%-30%A), 5~8 min (30%-36%A), 8~15 min (36%-48%A), 15~20 min (48%-70%A), 20~25 min (70%-90%A), 25~28 min (90%A), 28~34 min (90%-25%A).

[0051] Flow rate: 0.2 mL / min, using a conventional C18 chromatographic column (2.1 mm × 100 mm, 1.7 μm), column temperature 35 °C, injection volume 2 μL.

[0052] Mass spectrometry conditions: The mass spectrometers were equipped with an electrospray ionization source (ESI). Negative ion scanning mode: capillary temperature 320°C, sheath gas 35 arb unit, auxiliary gas 10 arb unit, sweep gas 30 arb unit, capillary voltage -3200V.

[0053] Figure 3 This is the total ion flow diagram of ginsenoside Rb1 after 30 minutes of conversion under the catalysis of nano zero-valent iron particles of Sophora japonica extract. The conversion process revealed four unknown peaks, which were marked as peaks 1 to 4 according to the retention time. Compounds 3 and 4 appeared after 5 minutes of reaction, and compounds 1 and 2 appeared over time. As the reaction proceeded, the content of compounds 3 and 4 gradually decreased, and after 2 hours, ginsenoside Rb1 was completely converted into compounds 1 and 2. This shows that the synthesized nZVI can specifically convert ginsenoside Rb1 into other ginsenosides.

[0054] Figure 4 This is the secondary tandem spectrum of compound 1 in the total ion flow chart after 30 minutes of conversion of ginsenoside Rb1. According to [M+HCOO] - and [MH] -ions, the relative molecular mass of compounds 1 and 2 was calculated to be 802.43. The fragmentation patterns of peaks 1 and 2 in the tandem mass spectrometry are the same, indicating that peaks 1 and 2 are isomers. [MH] - ( m / z 801) and ginsenoside Rb1 ( m / z 1107) has a molecular weight difference of 306, indicating that peaks 1 and 2 may be the products of deglycosylation of ginsenoside Rb1 at position C20. As shown in the secondary tandem mass spectrometry, m / z The fragment ion at 639.66 is m / z The product of removing the glucose substituent from the parent ion of 801.43. m / z The difference between 477.52 and the characteristic [MH]- of diol aglycones is 18, indicating that hydration occurred at the double bond between C24 and C25 of the aglycones in peaks 1 and 2. In addition, m / z 477.52 and [MH] - The difference in molecular weight was 324, indicating that the two glucose substituents at the C3 position were retained in the product. Therefore, peaks 1 and 2 were identified as diastereomers of 25-OH-Rg3. Based on the relative polarity of ginsenoside diastereomers, peaks 1 and 2 were 20(S)-25-OH-Rg3 and 20(R)-25-OH-Rg3, respectively.

[0055] In order to further illustrate the influence of the specific preparation parameters of the present invention on the catalytic effect of the nano zero-valent iron catalyst, the following examples are used for illustration.

[0056] Example 1

[0057] The preparation method of the nano zero-valent iron catalyst comprises the following steps:

[0058] S1, take 20 g of Sophora japonica powder and add 400 mL of 70% ethanol into a reaction vessel, heat to 80°C, reflux for 1 hour, and then filter the mixed solution to obtain Sophora japonica extract;

[0059] S2. Take 100 mL of the prepared Sophora japonica extract, add 2.7 g of ferric chloride hexahydrate and place it in a reaction container. After hydrothermal synthesis for 3 h, centrifuge at 10,000 rpm for 10 min, wash with water and ethanol 4 times, and place in a vacuum drying oven at 60°C overnight to obtain nano zero-valent iron particles.

[0060] 20 mg of the prepared nano zero-valent iron particles were placed in a 40 ppm ginsenoside Rb1 solution and heated to 80°C in a water bath shaker for 120 min. After the reaction was completed, the reaction solution was placed at room temperature and filtered through a filter membrane to obtain a mixture, which was then analyzed by HPLC-MS. The conversion rate of ginsenoside Rb1 was 100%, and the proportion of 25-OH-Rg3 derivatives in the ginsenoside Rb1 conversion product (i.e., the selectivity of 25-OH-Rg3 derivatives) was 100%.

[0061] Example 2

[0062] The preparation method of the nano zero-valent iron catalyst is the same as that in Example 1. Take 20 mg of the prepared nano zero-valent iron particles and place them in a 40 ppm ginsenoside Rb1 solution, and heat the reaction at 80°C in a water bath shaker for 90 minutes. After the reaction is completed, the reaction solution is placed at room temperature and filtered through a filter membrane to obtain a mixture, which is then analyzed by HPLC-MS. The conversion rate of ginsenoside Rb1 is 97.19%, and the selectivity of 25-OH-Rg3 derivatives is 100%.

[0063] Example 3

[0064] The preparation method of the nano zero-valent iron catalyst is the same as that of Example 1. Take 20 mg of the prepared nano zero-valent iron particles and place them in a 40 ppm ginsenoside Rb1 solution, and heat them at 80°C in a water bath shaker for 60 minutes. After the reaction is completed, the reaction solution is placed at room temperature and filtered through a filter membrane to obtain a mixture, which is then analyzed by HPLC-MS. The conversion rate of ginsenoside Rb1 is 92.26%, and the selectivity of 25-OH-Rg3 derivatives is 100%.

[0065] Example 4

[0066] The preparation method of the nano zero-valent iron catalyst is the same as that in Example 1. 20 mg of the prepared nano zero-valent iron particles are placed in a 40 ppm ginsenoside Rb1 solution, and heated to 80°C in a water bath shaker for 30 minutes. After the reaction is completed, the reaction solution is placed at room temperature and filtered through a filter membrane to obtain a mixture, which is then analyzed by HPLC-MS. The conversion rate of ginsenoside Rb1 is 73.39%, and the selectivity of 25-OH-Rg3 derivatives is 43.82%.

[0067] Example 5

[0068] The preparation method of the nano zero-valent iron catalyst is the same as that in Example 1. Take 20 mg of the prepared nano zero-valent iron particles and place them in a 40 ppm ginsenoside Rb1 solution, and heat them at 80°C in a water bath shaker for 5 minutes. After the reaction is completed, the reaction solution is placed at room temperature and filtered through a filter membrane to obtain a mixture, which is then analyzed by HPLC-MS. The conversion rate of ginsenoside Rb1 is 36.43%, and the selectivity of 25-OH-Rg3 derivatives is 35.71%.

[0069] Example 6

[0070] The preparation method of the nano zero-valent iron catalyst is the same as that of Example 1. 10 mg of the prepared nano zero-valent iron particles are placed in a 40 ppm ginsenoside Rb1 solution, and heated to 80°C in a water bath shaker for 120 min. After the reaction is completed, the reaction solution is placed at room temperature and filtered through a filter membrane to obtain a mixture, which is then analyzed by HPLC-MS. The conversion rate of ginsenoside Rb1 is 100%, and the selectivity of 25-OH-Rg3 derivatives is 100%.

[0071] Example 7

[0072] The preparation method of the nano zero-valent iron catalyst is the same as that of Example 1. 10 mg of the prepared nano zero-valent iron particles are placed in a 40 ppm ginsenoside Rb1 solution, and heated to 80°C in a water bath shaker for 90 minutes. After the reaction is completed, the reaction solution is placed at room temperature and filtered through a filter membrane to obtain a mixture, which is then analyzed by HPLC-MS. The conversion rate of ginsenoside Rb1 is 97.02%, and the selectivity of 25-OH-Rg3 derivatives is 95.71%.

[0073] Example 8

[0074] The preparation method of the nano zero-valent iron catalyst is the same as that in Example 1. 10 mg of the prepared nano zero-valent iron particles are placed in a 40 ppm ginsenoside Rb1 solution, and heated to 80° C. in a water bath shaker for 60 min. After the reaction is completed, the reaction solution is placed at room temperature and filtered through a filter membrane to obtain a mixture, which is then analyzed by HPLC-MS. The conversion rate of ginsenoside Rb1 is 92.49%, and the selectivity of 25-OH-Rg3 derivatives is 89.71%.

[0075] Example 9

[0076] The preparation method of the nano zero-valent iron catalyst is the same as that of Example 1. 10 mg of the prepared nano zero-valent iron particles are placed in a 40 ppm ginsenoside Rb1 solution, and heated to 80°C in a water bath shaker for 30 minutes. After the reaction is completed, the reaction solution is placed at room temperature and filtered through a filter membrane to obtain a mixture, which is then analyzed by HPLC-MS. The conversion rate of ginsenoside Rb1 is 68.56%, and the selectivity of 25-OH-Rg3 derivatives is 0%.

[0077] Example 10

[0078] The preparation method of the nano zero-valent iron catalyst is the same as that of Example 1. 10 mg of the prepared nano zero-valent iron particles are placed in a 40 ppm ginsenoside Rb1 solution, and heated to 80°C in a water bath shaker for 5 minutes. After the reaction is completed, the reaction solution is placed at room temperature and filtered through a filter membrane to obtain a mixture, which is then analyzed by HPLC-MS. The conversion rate of ginsenoside Rb1 is 23.12%, and the selectivity of 25-OH-Rg3 derivatives is 0%.

[0079] Embodiment 11

[0080] The preparation method of the nano zero-valent iron catalyst is the same as that of Example 1. 5 mg of the prepared nano zero-valent iron particles are placed in a 40 ppm ginsenoside Rb1 solution, and the mixture is heated at 80° C. in a water bath shaker for 120 min. After the reaction is completed, the reaction solution is placed at room temperature and filtered through a filter membrane to obtain a mixture, which is then analyzed by HPLC-MS. The conversion rate of ginsenoside Rb1 is 81.09%, and the selectivity of 25-OH-Rg3 derivatives is 100%.

[0081] Example 12

[0082] The preparation method of the nano zero-valent iron catalyst is the same as that of Example 1. 5 mg of the prepared nano zero-valent iron particles are placed in a 40 ppm ginsenoside Rb1 solution, and heated to 80° C. in a water bath shaker for 90 min. After the reaction is completed, the reaction solution is placed at room temperature and filtered through a filter membrane to obtain a mixture, which is then analyzed by HPLC-MS. The conversion rate of ginsenoside Rb1 is 73.39%, and the selectivity of 25-OH-Rg3 derivatives is 75.81%.

[0083] Embodiment 13

[0084] The preparation method of the nano zero-valent iron catalyst is the same as that of Example 1. 5 mg of the prepared nano zero-valent iron particles are placed in a 40 ppm ginsenoside Rb1 solution, and heated to 80° C. in a water bath shaker for 60 min. After the reaction is completed, the reaction solution is placed at room temperature and filtered through a filter membrane to obtain a mixture, which is then analyzed by HPLC-MS. The conversion rate of ginsenoside Rb1 is 43.02%, and the selectivity of 25-OH-Rg3 derivatives is 67.11%.

[0085] Embodiment 14

[0086] The preparation method of the nano zero-valent iron catalyst is the same as that in Example 1. 5 mg of the prepared nano zero-valent iron particles are placed in a 40 ppm ginsenoside Rb1 solution, and heated to 80° C. in a water bath shaker for 30 min. After the reaction is completed, the reaction solution is placed at room temperature and filtered through a filter membrane to obtain a mixture, which is then analyzed by HPLC-MS. The conversion rate of ginsenoside Rb1 is 22.11%, and the selectivity of 25-OH-Rg3 derivatives is 0%.

[0087] Embodiment 15

[0088] The preparation method of the nano zero-valent iron catalyst is the same as that in Example 1. 5 mg of the prepared nano zero-valent iron particles are placed in a 40 ppm ginsenoside Rb1 solution, and heated to 80° C. in a water bath shaker for 5 min. After the reaction is completed, the reaction solution is placed at room temperature and filtered through a filter membrane to obtain a mixture, which is then analyzed by HPLC-MS. The conversion rate of ginsenoside Rb1 is 10.80%, and the selectivity of 25-OH-Rg3 derivatives is 0%.

[0089] Comparative Example 1

[0090] The preparation method of the nano zero-valent iron catalyst comprises the following steps:

[0091] S1. Take 20 g of astragalus powder and add 400 mL of 70% ethanol into a reaction container. Heat to 80°C and reflux for 1 hour. Then, filter the mixed solution to obtain an astragalus extract.

[0092] S2. Take 100 mL of the prepared astragalus extract, add 2.7 g of ferric chloride hexahydrate and place it in a reaction container. After hydrothermal synthesis for 3 hours, centrifuge at 10,000 rpm for 10 minutes, wash with water and ethanol four times, and place in a vacuum drying oven at 60°C overnight to obtain nano zero-valent iron particles.

[0093] 10 mg of the prepared nano zero-valent iron particles were placed in a 40 ppm ginsenoside Rb1 solution and heated at 80°C in a water bath shaker for 120 min. After the reaction was completed, the reaction solution was placed at room temperature and filtered through a filter membrane to obtain a mixture, which was then analyzed by HPLC-MS. The conversion rate of ginsenoside Rb1 was 55.41%, and the selectivity of 25-OH-Rg3 derivatives was 34.37%.

[0094] Comparative Example 2

[0095] The preparation method of the nano zero-valent iron catalyst comprises the following steps:

[0096] S1. Take 20 g of honeysuckle powder and add 400 mL of 70% ethanol into a reaction container, heat to 80°C, reflux for 1 hour, and then filter the mixed solution to obtain a honeysuckle extract;

[0097] S2. Take 100 mL of the prepared honeysuckle extract, add 2.7 g of ferric chloride hexahydrate and place it in a reaction container. After hydrothermal synthesis for 3 hours, centrifuge at 10,000 rpm for 10 minutes, wash with water and ethanol four times, and place in a vacuum drying oven at 60°C overnight to obtain nano zero-valent iron particles.

[0098] 10 mg of the prepared nano zero-valent iron particles were placed in a 40 ppm ginsenoside Rb1 solution and heated at 80°C in a water bath shaker for 120 min. After the reaction was completed, the reaction solution was placed at room temperature and filtered through a filter membrane to obtain a mixture, which was then analyzed by HPLC-MS. The conversion rate of ginsenoside Rb1 was 44.62%, and the selectivity of 25-OH-Rg3 derivatives was 35.01%.

[0099] Comparative Example 3

[0100] The preparation method of the nano zero-valent iron catalyst comprises the following steps:

[0101] S1, take 20 g of Albizzia julibrissin powder and add 400 mL of 70% ethanol into a reaction vessel, heat to 80°C, reflux for 1 hour, and then filter the mixed solution to obtain Albizzia julibrissin extract;

[0102] S2. Take 100 mL of the prepared Albizzia julibrissin flower extract, add 2.7 g of ferric chloride hexahydrate and place it in a reaction container. After hydrothermal synthesis for 3 h, centrifuge at 10,000 rpm for 10 min, wash with water and ethanol 4 times, and place in a vacuum drying oven at 60°C overnight to obtain nano zero-valent iron particles.

[0103] 10 mg of the prepared nano zero-valent iron particles were placed in a 40 ppm ginsenoside Rb1 solution and heated in a water bath shaker at 80°C for 120 min. After the reaction was completed, the reaction solution was placed at room temperature and filtered through a filter membrane to obtain a mixture, which was then analyzed by HPLC-MS. The conversion rate of ginsenoside Rb1 was 22.75%, and the selectivity of 25-OH-Rg3 derivatives was 25.76%.

[0104] Table 1 Relationship between the dosages of ginsenoside Rb1 used to catalyze the conversion of 25-OH-Rg3 derivatives in the examples and comparative examples

[0105] Nano zero-valent iron particle dosage / mg Reaction time / min Ginsenoside Rb1 conversion rate Selectivity of 25-OH-Rg3 derivatives Example 1 20 120 100% 100% Example 2 20 90 97.19% 100% Example 3 20 60 92.26% 100% Example 4 20 30 73.39% 43.82% Example 5 20 5 36.43％ 35.71％ Example 6 10 120 100% 100% Example 7 10 90 97.02% 95.71% Example 8 10 60 92.49% 89.71% Example 9 10 30 68.56% 0% Example 10 10 5 23.12% 0% Embodiment 11 5 120 81.09% 100% Example 12 5 90 73.39% 75.81% Embodiment 13 5 60 43.02% 67.11% Embodiment 14 5 30 22.11% 0% Embodiment 15 5 5 10.8% 0% Comparative Example 1 10 120 55.41% 34.37% Comparative Example 2 10 120 44.62% 35.01% Comparative Example 3 10 120 22.75% 25.76%

[0106] It can be seen from the above examples and Table 1 that the activity of the nano zero-valent iron catalyst prepared from Sophora japonica extract in catalyzing the conversion of ginsenoside Rb1 is related to the dosage and reaction time. It can be seen from Examples 1-5 that the 25-OH-Rg3 derivative is generated later in the conversion of ginsenoside Rb1 and is greatly affected by the reaction time. When the reaction time is less than 30 minutes, the selectivity of the 25-OH-Rg3 derivative is low. In particular, when the nano zero-valent iron catalyst is less, the selectivity is affected more. For example, in Examples 9, 10, 14, and 15, the 25-OH-Rg3 derivative has not been generated. However, when the reaction time is sufficient, no matter how much catalyst is used, the reaction is about close to 120 minutes, the closer the ginsenoside Rb1 conversion rate is to 100%, the higher the selectivity of the 25-OH-Rg3 derivative.

[0107] The difference between Comparative Examples 1-3 and Example 6 is that the raw materials for preparing nano zero-valent iron are different. Plant extracts made of Astragalus, Honeysuckle, and Albizzia julibrissin are used to reduce and produce nano zero-valent iron. Although from the perspective of catalyst generation, green plant extracts can mostly generate nano zero-valent iron. However, when the product is used for the catalytic reaction of ginsenoside Rb1, it is found that the conversion rate of ginsenoside Rb1 varies greatly, and the selectivity of the obtained 25-OH-Rg3 derivatives also varies greatly; as shown in Tables 1 and Figure 5 As shown in the figure, the nano zero-valent iron catalyst prepared by Sophora japonica extract has a very high catalytic efficiency, which can make the conversion rate of ginsenoside Rb1 100%. The selectivity of 25-OH-Rg3 derivatives in the conversion products is also the strongest; the nano zero-valent iron catalyst prepared by Astragalus extract and honeysuckle extract has the second highest conversion rate of ginsenoside Rb1, with a conversion rate of 45%~55%, among which the selectivity of 25-OH-Rg3 derivatives is relatively low; the nano zero-valent iron catalyst prepared by Albizzia julibrissin extract has the worst conversion rate of ginsenoside Rb1, with a conversion rate of 22.75%, and the selectivity of 25-OH-Rg3 derivatives is the worst, at 25.76%.

[0108] The reason may be that the active ingredients such as flavonoids, flavonoids and polyphenols in Sophora japonica are relatively high. When used in the reduction preparation of nano zero-valent iron, they have a stronger ability to reduce trivalent iron ions and reduce the degree of agglomeration of nano zero-valent iron particles, increasing the specific surface area of ​​nano zero-valent iron, leading to an increase in catalytic conversion rate; it may also be due to the influence of the remaining flavonoids and other active ingredients in the Sophora japonica extract on the selectivity of the catalytic process of the nano zero-valent iron catalyst. The specific reason still needs further study; but the nano zero-valent iron catalyst prepared by Sophora japonica extract has a high specificity for the conversion of ginsenoside Rb1 to Rg3 derivatives, which is much higher than the existing reported conversion rate. It is worth mentioning that in the catalytic reaction, ginsenoside Rb1 is not converted into rare ginsenoside Rg3, but basically converted into 25-OH-Rg3 derivatives. According to previous studies, the hydroxyl substituent at the 25th position of Rg3 is mostly derived from water molecules in the ginsenoside Rb1 solution.

[0109] Generally speaking, derivatives of rare ginsenosides will also have the same medicinal properties as the corresponding rare ginsenosides. However, in order to further confirm the medicinal value of 25-OH-Rg3 derivatives, the activity test of rare ginsenoside Rg3 in lowering uric acid, which has been reported in combination with other rare ginsenosides Rk1, Rg6 and Rg5, was used as an example to test the effect of 25-OH-Rg3 derivatives alone on the activity of uric acid.

[0110] C57BL / 6 male mice (25-30g) were selected and fed for 1 week under constant temperature, constant humidity, 12h light / dark cycle, free access to food and water. All mice were randomly divided into 6 groups, 6 mice in each group. Except for the blank group, the other groups were gavaged with 0.5% sodium carboxymethylcellulose suspension of hypoxanthine (500 mg / kg / d) and potassium oxonate (500 mg / kg / d) once a day to establish a hyperuricemia mouse model. After 2 weeks, the serum uric acid content of mice increased by 2 times stably and began to be gavaged with drugs. The specific dosing schedule was as follows: the blank group (Control) and the model group (Model) were gavaged with the same amount of normal saline, the positive control group (Allopurinol) was gavaged with 15 mg / kg / d of allopurinol, and the low, medium and high dose groups of 25-OH-Rg3 derivatives (25-OH-Rg3-L, 25-OH-Rg3-M, 25-OH-Rg3-H) were gavaged with 25-OH-Rg3 derivatives at 25, 50 and 100 mg / kg / d, respectively, for 2 consecutive weeks. The mice were fasted but not watered 12 hours before the end of the experiment, and blood, liver and kidney samples were collected. After the blood samples were allowed to stand at room temperature for 1 hour, they were centrifuged at 3000 r / min at 4℃ for 10 minutes to obtain serum; for liver and kidney tissue samples, connective tissue, adipose tissue and hair were removed, and they were quickly frozen in liquid nitrogen for 0.5 hours. After processing, the above samples were transferred to -80℃ for medium- and long-term storage.

[0111] The levels of uric acid (UA), creatinine (Cr), and urea nitrogen (BUN) in the serum of each group of mice were determined according to the instructions of the biochemical detection kit. Figure 6 , Figure 7 , Figure 8 As shown ( n = 6). Figure 6 is the serum uric acid level of mice, vs Control, #### p <0.0001, vs Model, **** p <0.0001, *** p <0.001.

[0112] Uric acid level is a direct indicator of hyperuricemia. Figure 6 It can be seen that under the combined action of supplementing uric acid precursor (hypoxanthine) and uricase inhibitor (potassium oxonate), the serum uric acid level of mice in the model group (Model) was significantly higher than that in the blank group (Control), indicating that the mouse model of hyperuricemia was successfully constructed; the positive control drug can significantly reduce the serum uric acid level of hyperuricemia mice; the low, medium and high dose administration groups of 25-OH-Rg3 derivatives can reduce the serum UA level of hyperuricemia mice, and show an obvious dose-dependent relationship, indicating that 25-OH-Rg3 derivatives have the effect of reducing the serum uric acid level of hyperuricemia mice.

[0113] Figure 7 Serum creatinine level in mice, Figure 8 is the level of urea nitrogen in mouse serum, vs Control, ### p <0.001, ## p <0.01, vs Model, *** p <0.001, ** p <0.01, * p <0.05. Long-term accumulation of urate in the kidneys can cause kidney damage. Creatinine and urea nitrogen are important indicators of kidney damage. Figure 7 It can be seen that the serum creatinine level in the model group was higher than that in the blank group, and the medium and high dose groups of 25-OH-Rg3 derivatives could reduce the serum creatinine level. Figure 8 It can be seen that the serum urea nitrogen level in the model group was significantly higher than that in the blank group, and the positive control group and the medium and high dose groups of 25-OH-Rg3 derivatives could reduce the serum urea nitrogen level.

[0114] Based on the above analysis results, it can be preliminarily judged that 25-OH-Rg3 derivatives have the effect of improving kidney damage and have an obvious dose-effect relationship.

[0115] Weigh the mouse liver tissue accurately, add 0.9% physiological saline at a ratio of weight (g): volume (mL) = 1:9, homogenize in a cryogenic grinder to prepare a 10% homogenate, centrifuge at 3000 rpm for 10 min, take the supernatant, and determine the activity of xanthine oxidase (XOD) in the liver tissue according to the instructions of the biochemical detection kit. The results are shown in Fig. 9 ( n = 6). vs Control, #### p <0.0001, vs Model, **** p <0.0001, *** p <0.001, * p <0.05.

[0116] Compared with the blank group, the activity of xanthine oxidase in the liver of mice in the model group was significantly increased; allopurinol, as a xanthine oxidase inhibitor, has a good effect in inhibiting the activity of xanthine oxidase, and the experimental results also confirmed this. The activity of xanthine oxidase in the liver of the positive control group was significantly lower than that of the model group; the low, medium and high dose groups of 25-OH-Rg3 derivatives can inhibit the activity of xanthine oxidase in the liver of hyperuricemia mice, and are positively correlated with the dose, indicating that 25-OH-Rg3 derivatives have the effect of inhibiting UA synthesis by reducing the activity of xanthine oxidase.

[0117] Continue to weigh the mouse kidney tissue, cut it into pieces with clean scissors, and take an appropriate amount and place it in a 2 mL grinding tube. Add the protease inhibitor mixture (universal type, 50×), phosphatase inhibitor mixture (50×) and 0.05 M EDTA to the lysate at a ratio of 1:50. Add zirconium beads to the grinding tube, then add 10 times the amount of lysate containing protease inhibitors, and grind it in a low-temperature tissue grinder. After grinding evenly, centrifuge at 4°C, 12000 rpm, and take the supernatant and divide it into clean centrifuge tubes. Take the supernatant and calculate the total protein concentration of each group of samples according to the instructions of the BCA kit, adjust the total protein concentration of all samples to the same (5μg / μL), and make up for the insufficient amount with lysate. Mix the volume of protein loading buffer (5×) and protein sample in a ratio of 1:4, and heat and boil for 10 minutes to denature the protein. The Western-blot method was used to investigate the expression of related proteins in the kidney. The results are shown in the table. Fig.10 (n = 3).

[0118] 10a is the expression bar graph of related proteins in mouse kidney; 10b is the expression level of ABCG2; 10c is the expression level of GLUT9; 10d is the expression level of URAT1. vs Control, #### p <0.0001, ### p <0.001, vs Model, **** p <0.0001, *** p <0.001, ** p <0.01, * p <0.05.

[0119] ABCG2 is an important uric acid transporter in the kidney. If their function is abnormal, it may lead to reduced renal uric acid secretion and ultimately lead to renal excretion-deficient hyperuricemia. Fig.10 In b, compared with the blank group, the expression level of ABCG2 in the kidneys of mice in the model group was significantly decreased, the expression level of ABCG2 in the kidneys of mice in the positive control group was increased, and the expression level of ABCG2 in the medium and high dose groups of 25-OH-Rg3 derivatives gradually increased in a dose-dependent manner.

[0120] URAT1 and GLUT9 have extremely strong uric acid transport capabilities and can reabsorb uric acid in the lumen of the renal proximal tubules. They are important uric acid reabsorption transporters. Fig.10 c. Compared with the blank group, the expression levels of GLUT9 and URAT1 in the kidney of the model group mice were significantly increased at 10 days. Compared with the model group, the expression levels of URAT1 and GLUT9 in the kidney of the positive control group were different. Allopurinol could significantly reduce the expression of GLUT9, and had a certain regulatory effect on the expression of URAT1, but it was not statistically significant. The low, medium and high dose groups of 25-OH-Rg3 derivatives could significantly reduce the expression levels of GLUT9 and URAT1. At the same time, the regulatory effect of each dose group of 25-OH-Rg3 derivatives on GLUT9 was stronger than that on URAT1, and better than the positive control group.

[0121] In summary, the results of pharmacodynamic experiments showed that 25-OH-Rg3 derivatives can reduce the levels of uric acid, creatinine, and urea nitrogen in the serum of hyperuricemia mice and alleviate kidney damage; reduce the activity of xanthine oxidase in the liver and reduce uric acid synthesis; increase the expression of uric acid secretion protein ABCG2, reduce uric acid reabsorption proteins GLUT9 and URAT1, and promote uric acid excretion. The results show that 25-OH-Rg3 derivatives have the effects of lowering uric acid and protecting the kidneys.

[0122] The above is only a preferred embodiment of the present invention, and does not limit the present invention in any form. Although the present invention has been disclosed as a preferred embodiment, it is not used to limit the present invention. Any technician familiar with this patent can make some changes or modify the technical contents suggested above into equivalent embodiments without departing from the scope of the technical solution of the present invention. However, any simple modification, equivalent change and modification made to the above embodiments according to the technical essence of the present invention without departing from the content of the technical solution of the present invention still fall within the scope of the solution of the present invention.

Claims

1. Application of a nano zero-valent iron catalyst in the transformation of rare ginsenosides, characterized in that: Nano zero-valent iron catalyst is prepared by the following steps: S1, taking dried Sophora japonica powder and adding a certain proportion of ethanol solution into a reaction vessel, heating and refluxing for extraction for a certain period of time, and then filtering the mixed solution to obtain Sophora japonica extract; S2, adding a certain amount of ferric chloride hexahydrate to the prepared Sophora japonica extract in a container, performing hydrothermal synthesis for a certain period of time, performing centrifugal washing, and performing vacuum drying to obtain nano zero-valent iron particles; The nano zero-valent iron catalyst is used to convert ginsenoside Rb1 into a rare ginsenoside derivative 25-OH-Rg3; the specific application method includes: taking the prepared nano zero-valent iron particles and adding them to the ginsenoside Rb1 solution, heating and converting in a water bath shaker; After the reaction is completed, the reaction solution is placed at room temperature and then filtered through a filter membrane to obtain a 25-OH-Rg3 derivative; the mass ratio of nano zero-valent iron particles to ginsenoside Rb1 is 1:0.02-0.08; the concentration of ginsenoside Rb1 solution is 10~100ppm; the heating temperature of the water bath shaker is 20~80℃, and the conversion time is 60~180min.

2. The application of the nano zero-valent iron catalyst in the transformation of rare ginsenosides according to claim 1, characterized in that: The liquid-to-solid ratio of the dry Sophora japonica flower powder to the ethanol solution is 10:1-50:1; the concentration of the ethanol solution is 40%-100%.

3. The application of the nano zero-valent iron catalyst in the transformation of rare ginsenosides according to claim 1, characterized in that: The heating temperature is 50~90℃ and the reflux extraction time is 1~3h.

4. The use of the nano zero-valent iron catalyst in the conversion of rare ginsenosides according to claim 1, characterized in that: The mass ratio of ferric chloride hexahydrate to the volume ratio of Sophora japonica extract is 2.7 mg / mL-54.0 mg / mL.

5. The use of the nano zero-valent iron catalyst in the conversion of rare ginsenosides according to claim 2, characterized in that: The liquid-to-solid ratio of dried Sophora japonica seeds to the extraction solvent is 20:1, and the concentration of the ethanol solution is 70%.

6. The use of the nano zero-valent iron catalyst in the transformation of rare ginsenosides according to claim 3, characterized in that: The heating temperature was 80°C and the reflux extraction time was 1 h.

7. The use of the nano zero-valent iron catalyst in the conversion of rare ginsenosides according to claim 4, characterized in that: The mass ratio of ferric chloride hexahydrate to the volume ratio of Sophora japonica extract is 27 mg / mL.

8. The use of the nano zero-valent iron catalyst according to claim 7 in the conversion of rare ginsenosides, characterized in that: The concentration of ginsenoside Rb1 solution was 40 ppm, and the temperature of the water bath shaker was 80°C.

9. The use of the nano zero-valent iron catalyst in the transformation of rare ginsenosides according to claim 8, characterized in that: The conversion time was 120 min.

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