Chemically modified rare ginsenoside derivative, and preparation method and application thereof

Abstract

The present application belongs to the technical field of natural product chemical modification, and particularly relates to a chemically modified rare ginsenoside derivative, a preparation method and application thereof. The present application takes rare ginsenoside as raw material, introduces a sulfate group by sulfating modification on the rare ginsenoside to obtain a chemically modified rare ginsenoside derivative; the modifier used in the sulfating modification is a sulfonating agent. The rare ginsenoside is synthesized by conversion with ginsenoside Rb1 as raw material and AlCl3 as catalyst in an ethanol system. The modification method of the present application is simple in operation steps, does not require special equipment, is suitable for large-scale production, and has a broad application prospect in the field of drug preparation development and the like.

Description

Technical Field

[0001] This invention belongs to the field of natural product chemical modification technology, specifically relating to a chemically modified rare ginsenoside derivative, its preparation method, and its application. Background Technology

[0002] Ginsenosides are the main active components of ginseng, and their pharmacological activities have attracted much attention. In particular, rare ginsenosides such as Rg3 and Rh2 have garnered widespread interest due to their significant anti-tumor, anti-inflammatory, and immunomodulatory activities. However, naturally occurring rare ginsenosides are present in extremely low amounts in natural plants, and their extraction and separation are difficult and costly, severely limiting their in-depth research and clinical application.

[0003] Currently, existing technologies typically employ transformation methods to prepare rare ginsenosides, mainly including biological transformation and chemical transformation. However, these methods generally suffer from the following drawbacks: biological transformation is time-consuming, prone to microbial contamination, and difficult to control; while traditional chemical transformation involves harsh reaction conditions, poor selectivity, and easily damages the saponin nucleus structure, resulting in low yields of the target product and complex byproducts, making it difficult to meet the needs of large-scale, standardized production.

[0004] Chemical modification is an important means of improving the physicochemical properties and biological activity of natural products. Although existing technologies have attempted to derivatize ginsenosides—for example, patent application CN115651050A discloses a method for reacting ordinary ginsenosides with sodium bisulfite under heavy metal salt catalysis to generate sulfonic acid derivatives—this method has significant drawbacks: the reaction relies on heavy metal catalysts, which can easily introduce toxic residues, failing to meet the safety and environmental protection requirements of pharmaceutical production, and the subsequent purification steps are cumbersome. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention provides a chemically modified rare ginsenoside derivative, its preparation method, and its applications. This invention uses rare ginsenosides as a direct raw material and employs a sulfur trioxide-pyridine method to introduce sulfate groups onto the rare ginsenosides through an electrophilic substitution reaction. This improves the physicochemical properties of the rare ginsenosides, enhancing their bioavailability and activity stability. Furthermore, this method is simple to operate, produces high-purity products, and is suitable for industrial production. It also solves the technical problems of existing modification methods that rely on heavy metal catalysts, easily introduce toxic residues, and involve cumbersome subsequent purification steps.

[0006] The first objective of this invention is to provide a method for preparing chemically modified rare ginsenoside derivatives, comprising the following steps:

[0007] Using rare ginsenosides as raw materials, rare ginsenosides, sulfonating agents, and solvents undergo an electrophilic substitution reaction at 60℃~70℃ to introduce sulfate groups onto the rare ginsenosides, thereby obtaining chemically modified rare ginsenoside derivatives.

[0008] Preferably, the mass ratio of rare ginsenosides to sulfonating agent is 20:8~9.

[0009] Preferably, the sulfonating agent is a sulfur trioxide-pyridine complex.

[0010] Preferably, the ratio of rare ginsenosides to solvent is 100mg:1mL~3mL.

[0011] Preferably, the electrophilic substitution reaction takes 4 to 6 hours.

[0012] Preferably, the solvent is dimethyl sulfoxide.

[0013] Preferably, rare ginsenosides are synthesized from ginsenoside Rb1 as a raw material and AlCl3 as a catalyst in a pure ethanol system; the structural formula of ginsenoside Rb1 is as follows:

[0014] .

[0015] The preferred method for preparing rare ginsenosides is as follows:

[0016] Ginsenoside Rb1 and AlCl3 were mixed evenly in pure ethanol solvent, and then reacted at 45℃~50℃ for 24h. After purification, rare ginsenosides were obtained.

[0017] Preferred rare ginsenosides include Rg5, Rg3 and Rk1 products; the yields of Rg5, Rg3 and Rk1 are 68.57%, 6.55% and 2.98%, respectively.

[0018] A second objective of this invention is to provide chemically modified rare ginsenoside derivatives prepared by the above-described method.

[0019] A third objective of this invention is to provide the application of the above-mentioned chemically modified rare ginsenoside derivatives in the preparation of antitumor drugs.

[0020] Compared with the prior art, the present invention has the following technical effects:

[0021] This invention uses rare ginsenosides as direct raw materials, and involves an electrophilic substitution reaction of rare ginsenosides, a sulfonating agent, and a solvent at 60°C to 70°C to introduce sulfate groups onto the rare ginsenosides, thereby obtaining chemically modified rare ginsenoside derivatives. This process eliminates the need for heavy metal catalysts, thus avoiding the risk of toxic side effects from heavy metal residues and making the product more compliant with pharmaceutical safety standards.

[0022] The sulfonation modification method described in this invention features a moderate reaction temperature and controllable time. These mild conditions effectively avoid damage to the core structure of rare ginsenosides and excessive substitution of sulfate groups, ensuring high selectivity and structural stability of the product. Simultaneously, this method simplifies post-processing purification steps, avoiding complex purification operations for catalyst removal, making the process greener and more economical.

[0023] The chemically modified rare ginsenoside derivatives prepared in this invention exhibit significantly improved water solubility, cell permeability, and inhibitory activity against specific tumor cell lines compared to unmodified rare ginsenosides. This provides a novel and highly promising candidate compound for solving the problem of poor drug-likeness of rare ginsenosides, and opens up a new direction for the development of anti-tumor drugs. Attached Figure Description

[0024] Figure 1 The total ion chromatogram and mass spectrum of S-Rb1 prepared for Comparative Example 1 are shown below. (a) is the total ion chromatogram, (b) is the mass spectrum of substance 1 obtained by sulfation modification of S-Rb1, (c) is the mass spectrum of substance 2 obtained by sulfation modification of S-Rb1, and (d) is the mass spectrum of substance 3 obtained by sulfation modification of S-Rb1.

[0025] Figure 2 The total ion chromatogram and mass spectrum of S-Rg3 / 5 prepared in Example 1 are shown below. (a) is the total ion chromatogram, (b) is the mass spectrum of substance 1 obtained by sulfation modification of S-Rg3 / 5, (c) is the mass spectrum of substance 2 obtained by sulfation modification of S-Rg3 / 5, (d) is the mass spectrum of substance 3 obtained by sulfation modification of S-Rg3 / 5, (e) is the mass spectrum of substance 4 obtained by sulfation modification of S-Rg3 / 5, and (f) is the mass spectrum of substance 5 obtained by sulfation modification of S-Rg3 / 5.

[0026] Figure 3 In the study, (A) is the effect of ginsenoside Rb1 and rare ginsenoside Rg3 / 5 on the activity of MCF-7 cells, (B) is the effect of ginsenoside Rb1 and S-Rb1 on the activity of MCF-7 cells, (C) is the effect of S-Rb1 and S-Rg3 / 5 on the survival rate of MCF-7 cells, and (D) is the effect of Rg3 / 5 and S-Rg3 / 5 on the survival rate of MCF-7 cells.

[0027] Figure 4The effect of ginsenosides Rg3 / 5 and S-Rg3 / 5 on the migration activity of MCF-7 cells was detected by scratch assay. Among them, (A) shows the scratch migration map of cells in the S-Rg3 / 5 group, Rg3 / 5 group and control group; (B) shows the statistical results of the scratch migration rate of cells in the S-Rg3 / 5 group, Rg3 / 5 group and control group. Detailed Implementation

[0028] To enable those skilled in the art to better understand and implement the technical solutions of the present invention, the present invention will be further described below in conjunction with specific embodiments and accompanying drawings.

[0029] Unless otherwise specified, all reagents used in this invention are commercially available, and all methods used are conventional techniques in the art.

[0030] The following specific examples will provide further explanation.

[0031] It should be noted that the rare ginsenosides of this invention are synthesized using ginsenoside Rb1 as raw material and AlCl3 as catalyst, according to the method disclosed in the prior art: Optimization of AlCl3-catalyzed preparation of rare ginsenoside Rg5 [J]. Applied Chemical Industry, 2025, to obtain rare ginsenosides with higher biological activity; the specific synthetic route is as follows:

[0032] ;

[0033] Among them, rare ginsenosides include Rg5, Rg3 and Rk1 products; the yields of Rg5, Rg3 and Rk1 are 68.57%, 6.55% and 2.98% respectively, with Rg5 and Rg3 being the main products. This mixed product is denoted as rare ginsenoside Rg3 / 5.

[0034] Example 1

[0035] A method for preparing a chemically modified rare ginsenoside derivative includes the following steps:

[0036] 200 mg of rare ginsenoside Rg3 / 5 and 81 mg of sulfur trioxide-pyridine complex were sequentially added to 2 mL of dimethyl sulfoxide and mixed. The mixture was placed in a water bath at 65 °C and reacted for 4 h before cooling. 1 mol / L NaOH solution was added to adjust the mixture to neutral, and then dialyzed for 72 h using a dialysis bag with a molecular weight of 500 Da. The mixture was concentrated and lyophilized to obtain a chemically modified rare ginsenoside derivative, denoted as S-Rg3 / 5.

[0037] Example 2

[0038] A method for preparing a chemically modified rare ginsenoside derivative includes the following steps:

[0039] 200 mg of rare ginsenoside Rg3 / 5 and 90 mg of sulfur trioxide-pyridine complex were sequentially added to 6 mL of dimethyl sulfoxide and mixed. The mixture was placed in a water bath at 70 °C and reacted for 6 h before cooling. 1 mol / L NaOH solution was added to adjust the mixture to neutral, and then dialyzed for 72 h using a dialysis bag with a molecular weight of 500 Da. The mixture was concentrated and lyophilized to obtain a chemically modified rare ginsenoside derivative, denoted as S-Rg3 / 5.

[0040] Example 3

[0041] A method for preparing a chemically modified rare ginsenoside derivative includes the following steps:

[0042] 200 mg of rare ginsenoside Rg3 / 5 and 80 mg of sulfur trioxide-pyridine complex were sequentially added to 2 mL of dimethyl sulfoxide and mixed. The mixture was placed in a water bath at 60 °C and reacted for 4 h before cooling. 1 mol / L NaOH solution was added to adjust the mixture to neutral, and then dialyzed for 72 h using a dialysis bag with a molecular weight of 500 Da. The mixture was concentrated and lyophilized to obtain a chemically modified rare ginsenoside derivative, denoted as S-Rg3 / 5.

[0043] Comparative Example 1

[0044] 200 mg of ordinary ginsenoside Rb1 and 81 mg of sulfur trioxide-pyridine complex were sequentially added to 2 mL of dimethyl sulfoxide and mixed. The mixture was placed in a water bath at 65 °C and reacted for 4 h before cooling. 1 mol / L NaOH solution was added to adjust the mixture to neutral, and then dialyzed for 72 h using a dialysis bag with a molecular weight of 500 Da. The mixture was concentrated and lyophilized to obtain a chemically modified rare ginsenoside derivative, denoted as S-Rb1.

[0045] The chemically modified rare ginsenoside derivatives obtained in Examples 2-3 of this invention are used as examples for further research. The specific research methods and results are shown below:

[0046] 1. High performance liquid chromatography-mass spectrometry (HPLC-MS) testing.

[0047] like Figure 1 As shown in (a) of the diagram, the elution times of substances in S-Rb1 are mainly concentrated before 7 minutes, with three main characteristic peaks labeled as 1, 2, and 3. Figure 1 As shown in (b), substance 1 has a mass-to-charge ratio of 362.4 for [M+4(SO3)2]. - )] 4- This is because the hydroxyl group in the sugar group of ordinary ginsenoside Rb1 is introduced with -SO3⁻, giving the ginsenoside a negative charge; substance 1 introduces four sulfate groups into the sugar group of ordinary ginsenoside Rb1. For example... Figure 1As shown in (c), substance 2 has a mass-to-charge ratio of 447.8 for [M+3(SO3)2]. - )] 3- Substance 2 is formed by introducing three sulfate groups into the glycosyl group of ginsenoside Rb1. For example... Figure 1 As shown in (d), substance 3 has a mass-to-charge ratio of 594.8 in [MH]. + +SO3 - ] 2- Substance 3 is a substance in which a sulfate group is introduced into the glycosyl group of ginsenoside Rb1.

[0048] This shows that the water solubility of the sulfated product is improved, and the higher the degree of sulfation, the better the water solubility, which may enhance bioavailability. The introduction of negative charge may also change its absorption, distribution and metabolic characteristics in vivo.

[0049] like Figure 2 As shown in (a) of the image, the elution times of substances in S-Rg3 / 5 are mainly concentrated between 5 and 15 minutes, with five main characteristic peaks labeled as 1, 2, 3, 4, and 5. Figure 2 As shown in (b), substance 1 has a mass-to-charge ratio of 340.3 for [M+3SO3]. - )] 3- This is because three sulfate groups were introduced into the glycosyl group of rare ginsenoside Rg3. However, due to the instability of highly sulfated compounds, the sulfate groups were destroyed during mass spectrometry analysis, resulting in desulfation and producing [M+2SO3] with a mass-to-charge ratio of 471. - ] 2- ; Figure 2 As shown in (c), substance 2 has a mass-to-charge ratio of 334.3 [M+3(SO3)2]. - )] 3- Substance 2 is a rare ginsenoside Rg5 in which three sulfate groups are introduced into the glycosyl group; Figure 2 As shown in (d), substance 3 has a mass-to-charge ratio of 471 for [M+2SO3]. - ] 2- Substance 3 is a rare ginsenoside Rg3 in which two sulfate groups are introduced into the glycosyl group. For example... Figure 2 As shown in (e), substance 4 has a mass-to-charge ratio of 462.1 for [M+2SO3]. - ] 2- Substance 4 is a rare ginsenoside Rg5 in which two sulfate groups are introduced into the glycosyl group. Figure 2 As shown in (f), substance 5 has a mass-to-charge ratio of 845 for [M+SO3]. - ] - Substance 5 is a rare ginsenoside Rg5 in which a sulfate group is introduced into the glycosyl group.

[0050] Therefore, substance 1 is obtained by introducing three sulfate groups into the sugar group of Rg3, resulting in Rg3-3S; substance 2 is obtained by introducing three sulfate groups into the sugar group of Rg5, resulting in Rg5-3S; substance 3 is obtained by introducing two sulfate groups into the sugar group of Rg3, resulting in Rg3-2S; substance 4 is obtained by introducing two sulfate groups into the sugar group of Rg5, resulting in Rg5-2S; and substance 5 is obtained by introducing one sulfate group into the sugar group of Rg5, resulting in Rg5-S. The higher the degree of substitution of sulfate groups, the stronger the polarity of the product, which can effectively improve water solubility.

[0051] 2. MTT toxicity test.

[0052] Select MCF7 breast cancer cells with confluence exceeding 80%. Discard the culture supernatant, wash with 1 mL of PBS, and digest with 1 mL of trypsin (0.25% trypsin-EDTA) for 1 min. Observe the morphology under a microscope. Add 2 mL of complete culture medium to stop trypsin digestion, pipette the cells, transfer to centrifuge tubes, and centrifuge at 800 rpm for 5 min. Remove the supernatant after centrifugation. Add 4 mL of complete culture medium to gently resuspend the cells. While maintaining complete cell resuspension, take 10 μL and count the cells once using a cell counter. Calculate the dilution factor and dilute with complete culture medium. After dilution, continue counting with a cell counter to adjust the cell concentration to 2 × 10⁻⁶ cells / mL. 4 Cells / mL. Cells were seeded into groups, with 100 μL of diluted cell suspension added to each well of a 96-well plate. After incubation at 37°C and 5% CO2 for 24 h, the culture medium was discarded, and 100 μL of samples with different concentration gradients were added. A control group and a blank / zero group were set up, with the control group consisting of untreated cells and the blank / zero group containing no cells. After incubation at 37°C and 5% CO2 for another 24 h, 10 μL of 5 mg / mL MTT was added. After incubation at 37°C and 5% CO2 for another 4 h, the culture supernatant was discarded. 100 μL of DMSO was added to dissolve the cells, and the mixture was allowed to stand for 10 min. Cell viability was measured at 570 nm using a microplate reader, and cell viability was calculated according to the cell viability formula.

[0053] ;

[0054] In the formula: A1 is the absorbance of the experimental group; A0 is the absorbance of the blank zeroing group; A is the absorbance of the control group.

[0055] To investigate the inhibitory effect of ginsenosides on breast cancer activity, the MTT assay was used to determine the effect of different ginsenosides and their modified products on breast cancer activity at 24 h, and their 24-h half-inhibitory concentration (IC50) was determined. 50 As shown in Table 1, IC 50 The value can be used to measure the ability of a drug to induce apoptosis; the stronger the induction ability, the lower the value.

[0056] Table 1. IC50 of ginsenosides and their modified products on mammary gland cells after 24 hours of treatment. 50

[0057]

[0058] Table 1 shows that ordinary ginsenoside Rb1 has an effect on the IC50 of the breast cancer cell line MCF-7. 50 The value was 722.4 µg / mL, indicating that rare ginsenoside Rg3 / 5 had an IC50 value of 722.4 µg / mL against MCF-7 cells. 50 The value was 110.0 µg / mL, while the IC50 values ​​of S-Rb1 and S-Rg3 / 5 for MCF-7 cells were... 50 The values ​​were 235.0 µg / mL and 102.1 µg / mL, respectively. This means that for MCF-7 breast cancer cells, ordinary ginsenoside Rb1 had a weak inhibitory effect, while S-Rb1, rare ginsenoside Rg3 / 5, and S-Rg3 / 5 showed better inhibitory abilities.

[0059] like Figure 3 As shown in (A), Rb1 has a weak inhibitory effect on MCF-7 cells. At low concentrations, Rb1 and Rg3 / 5 showed no significant cytotoxicity to MCF-7 cells. At 400 µg / mL, the cell viability of the Rb1 group decreased to 76.03%, and that of the Rg3 / 5 group decreased to 3.46%, indicating that Rg3 / 5 has stronger cytotoxicity to MCF-7 cells. Figure 3 As shown in (B), at low drug concentrations, the cell survival rates after treatment with both Rb1 and S-Rb1 were close to 100%, with no significant difference (p>0.05). At concentrations greater than 50 µg / mL, the survival rates of the S-Rb1 group were lower than those of the Rb1 group. At a concentration of 200 µg / mL, the Rb1 group showed no significant cytotoxicity to MCF-7 cells, with a survival rate of 99.19%, while the survival rate of the S-Rb1 group was 76.03%, indicating that the modified S-Rb1 had a stronger inhibitory effect on breast cancer cells. With increasing ginsenoside concentration, the survival rate of MCF-7 cells gradually decreased, indicating that both ginsenoside Rb1 and its sulfated derivative S-Rb1 have inhibitory effects on breast cancer cells. Figure 3 As shown in (C), at 200 µg / mL, the cell viability of the S-Rb1 group was 76.03%, decreasing to 59.21% in the S-Rg3 / 5 group. At a concentration of 400 µg / mL, the MCF-7 cell viability was only 1.11%, indicating that S-Rg3 / 5 has stronger inhibitory activity against breast cancer than S-Rb1. Figure 3As shown in (D), both drugs exhibited dose-dependent inhibitory effects with increasing drug concentration. When the concentration reached 400 µg / mL, the survival rate of the Rg3 / 5 group was lower than that of the S-Rg3 / 5 group, demonstrating better cell inhibition.

[0060] 3. Migration capability detection.

[0061] Logarithmic growth phase MCF7 cells were seeded into 6-well plates with evenly drawn horizontal lines. Once the cells had reached confluence (over 90% confluence), a 200 μL pipette tip was used to make vertical scratches on the cell layer, parallel or perpendicular to the horizontal lines on the back. After scratching, the cells were washed 1-2 times with PBS to remove the scratched cells. 200 μg / mL of Rg3 / Rg5 and S-Rg3 / Rg5 basal medium were added, respectively. A control was prepared using basal medium without the drugs. Four replicates were set up. The horizontal lines drawn on the back of the 6-well plate were erased. Micrographs were taken at 0h, 24h, and 48h. The area of ​​the scratched area was calculated using ImageJ software, and the cell migration rate was calculated according to the formula.

[0062] ;

[0063] In the formula: S0 is the initial scratch area; S is the scratch area at each time point.

[0064] The effect of ginsenosides on the migration ability of breast cancer cells can be detected by cell scratch assay. In order to ensure that the vast majority of cells remain viable after the addition of the drug, a drug concentration of 200 μg / mL should be selected in the experiment.

[0065] like Figure 4 As shown, over time, the cells gradually filled the scratched area. At 24 h, the cell migration rate of the S-Rg3 / 5 group was 4.04%, significantly lower than that of the control group (28.28%) (P<0.0001), and the migration rate of the Rg3 / 5 group was 14.40%, also significantly lower than that of the control group (P<0.01). At 48 h, the cell migration rate of the S-Rg3 / 5 group was 6.44%, significantly lower than that of the control group (47.99%) (P<0.0001), and the migration rate of the Rg3 / 5 group was 17.29%, also significantly lower than that of the control group (P<0.0001). However, the effect of the S-Rg3 / 5 group was more prominent, showing a stronger inhibitory effect compared to Rg3 / 5 (P<0.05).

[0066] It should be noted that when numerical ranges are involved in this invention, it should be understood that both endpoints of each numerical range and any value between the two endpoints can be selected. Since the steps and methods used are the same as in the embodiments, preferred embodiments are described here to avoid redundancy. Although preferred embodiments of the invention have been described, those skilled in the art, once they understand the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments as well as all changes and modifications falling within the scope of this invention.

[0067] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. A method for preparing a chemically modified rare ginsenoside derivative, characterized in that, Includes the following steps: Using rare ginsenosides as raw materials, rare ginsenosides, sulfonating agents and solvents undergo an electrophilic substitution reaction at 60℃~70℃ to introduce sulfate groups onto rare ginsenosides, thereby obtaining chemically modified rare ginsenoside derivatives. The mass ratio of rare ginsenosides to sulfonating agent is 20:8~9; The sulfonating agent is a sulfur trioxide-pyridine complex; The electrophilic substitution reaction takes 4 to 6 hours. Rare ginsenosides are synthesized from ginsenoside Rb1 as a raw material, with AlCl3 as a catalyst, in an ethanol system at 45℃~50℃. The structural formula of ginsenoside Rb1 is as follows: ； The rare ginsenosides include Rg5, Rg3 and Rk1.

2. The method for preparing chemically modified rare ginsenoside derivatives according to claim 1, characterized in that, The ratio of rare ginsenosides to solvent is 100mg:1mL~3mL.

3. The method for preparing chemically modified rare ginsenoside derivatives according to claim 1, characterized in that, The solvent is dimethyl sulfoxide.

4. A chemically modified rare ginsenoside derivative, characterized in that, The chemically modified rare ginsenoside derivatives are prepared by the method described in any one of claims 1 to 3.

5. The application of a chemically modified rare ginsenoside derivative in the preparation of antitumor drugs, characterized in that, The chemically modified rare ginsenoside derivative is the chemically modified rare ginsenoside derivative as described in claim 4.

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