A method for rapid separation of ginsenoside Rd from total ginsenosides

Abstract

This invention relates to the field of natural product separation and purification technology, specifically to a method for rapidly separating ginsenoside Rd from total ginsenosides. The method includes the following steps: (1) preparation of zein solution; (2) dissolving total ginsenosides in zein solution; (3) preparing a nanoparticle solid dispersion by antisolvent precipitation; (4) removing ethanol from the nanoparticle dispersion by rotary evaporation; (5) filtering the nanoparticle dispersion using a filter membrane with a pore size smaller than the nanoparticle size; (6) pre-freezing and then freeze-drying the purified nanoparticle dispersion; and (7) extracting the freeze-dried particles with acetonitrile. This invention uses zein nanoparticles as a solid-phase extraction carrier to achieve highly selective recognition and enrichment of Rd during antisolvent self-assembly. By dissolving total ginsenosides and zein together, Rd can be separated simultaneously through a single antisolvent precipitation, simplifying the operation process.

Description

Technical Field

[0001] This invention relates to the field of natural product separation and purification technology, specifically to a method for rapidly separating ginsenoside Rd from total ginsenosides. Background Technology

[0002] Ginsenoside Rd is one of the main active monomers of precious traditional Chinese medicines such as ginseng and Panax notoginseng, possessing various pharmacological functions including neuroprotection, anti-inflammation, anti-tumor activity, and cardiovascular protection. In particular, two randomized controlled clinical trials have confirmed that ginsenoside Rd is safe and effective against acute ischemic stroke, demonstrating good potential for new drug development. However, both pharmacological research and formulation development require high-purity ginsenoside Rd monomers. Therefore, the efficient and low-cost isolation and purification of ginsenoside Rd from total ginsenosides is a prerequisite and key to promoting in-depth research on its efficacy and industrial application.

[0003] Currently, the mainstream techniques for isolating ginsenoside Rd monomers from total ginsenosides are column chromatography and preparative high-performance liquid chromatography (HPLC). These methods utilize the difference in retention time between Rd and other saponin monomers on the stationary phase for separation. While they can yield Rd products with a purity ≥98%, the procedures are cumbersome, the separation cycle is long, and the batch throughput is small, typically only in the milligram to gram range. Furthermore, they consume large amounts of organic solvents such as acetonitrile and methanol, and the chromatographic packing materials are expensive and have limited lifespans, resulting in high production costs and environmental pollution. The overall yield from total ginsenosides to Rd monomers is often less than 5%, making the market price of Rd monomers as high as several thousand to tens of thousands of yuan per gram. This limits their application to laboratory-scale research and fails to meet the needs of industrial production, severely restricting cost control and clinical application of Rd-related formulations.

[0004] To address the aforementioned problems, this invention proposes a novel purification approach: utilizing zein nanoparticles as a solid-phase extraction carrier, directly adding them to a mixed solution of total ginsenosides. By controlling the antisolvent self-assembly conditions, the zein nanoparticles selectively "capture" ginsenoside Rd from the complex saponin mixture, forming Rd-specifically encapsulated zein nanoparticles. A simple extraction then yields high-purity Rd monomers. This method eliminates the need for traditional column chromatography or preparative liquid chromatography, achieving selective enrichment in one step. It directly uses readily available and inexpensive total ginsenosides as raw materials, featuring a simple process, short cycle time, and low organic solvent consumption (mainly ethanol and water), making it suitable for large-scale production. Based on the solid-phase selective Rd capture technology using zein nanoparticles, this invention provides an economical, efficient, and industrially scalable new solution for the purification of ginsenoside Rd, potentially solving the problems of high cost and low efficiency of existing purification methods. This has significant value in promoting the drug development and clinical application of ginsenoside Rd.

[0005] Furthermore, on the one hand, there are differences in understanding among those skilled in the art; on the other hand, the inventors studied a large number of documents and patents when making this invention, but due to space limitations, not all details and contents were listed in detail. However, this does not mean that the present invention does not possess the features of these prior art. On the contrary, the present invention already possesses all the features of the prior art, and the applicant reserves the right to add relevant prior art to the background art. Summary of the Invention

[0006] This invention relates to the field of natural product separation and purification technology, specifically to a method for rapidly separating ginsenoside Rd from total ginsenosides.

[0007] The purpose of this invention is to provide a method for separating and purifying ginsenoside Rd from total ginsenosides, in order to solve the technical problems of existing column chromatography and preparative high-performance liquid chromatography methods, such as cumbersome operation procedures, long separation cycles, small batch throughput, large consumption of organic solvents, expensive chromatographic packing materials, total yield from total ginsenosides to Rd monomer less than 5%, high production costs, and environmental unfriendliness.

[0008] To address the aforementioned technical problems, one objective of this invention is to provide a method for rapidly separating ginsenoside Rd from total ginsenosides, comprising the following steps: (1) Dissolve zein in an ethanol solution to obtain a zein solution; (2) Dissolve total ginsenosides in zein solution to obtain a mixture; (3) The mixture is slowly injected into water, and under stirring conditions, the zein is triggered to self-assemble into nanoparticles, while ginsenoside Rd is selectively encapsulated to obtain a nanoparticle dispersion. (4) Ethanol in the nanoparticle dispersion was removed by rotary evaporation; (5) The nanoparticle dispersion was filtered by a filter membrane with a pore size smaller than that of the nanoparticles to retain the zein nanoparticles containing ginsenoside Rd. The nanoparticles had a particle size of 100~200 nm. Unencapsulated free ginsenosides were removed to obtain a purified nanoparticle dispersion. (6) The purified nanoparticle dispersion was pre-frozen and then freeze-dried to obtain zein freeze-dried particles containing ginsenoside Rd. (7) The freeze-dried particles were ultrasonically extracted with acetonitrile, and the supernatant was collected after centrifugation to obtain ginsenoside Rd with a purity ≥95%.

[0009] According to a preferred embodiment, in step (1), the volume fraction of the ethanol solution is 60%~80%. Preferably, in step (1), the volume fraction of the ethanol solution is 70%. Alternatively, the volume fraction of the ethanol solution is 60%. Or, the volume fraction of the ethanol solution is 80%.

[0010] According to a preferred embodiment, in step (2), the mass ratio of zein to total ginsenosides is 0.5~2:1. Preferably, in step (2), the mass ratio of zein to total ginsenosides is 1:1. The mass ratio of zein to total ginsenosides is 0.5:1. The mass ratio of zein to total ginsenosides is 2:1.

[0011] According to a preferred embodiment, in step (3), the volume ratio of the mixture to water is 1:3 to 6. Preferably, the volume ratio of the mixture to water is 1:3. The volume ratio of the mixture to water is 1:4. The volume ratio of the mixture to water is 1:5. The volume ratio of the mixture to water is 1:6.

[0012] According to a preferred embodiment, in step (4), the temperature of rotary evaporation is 40~50°C.

[0013] According to a preferred embodiment, in step (5), the pore size of the filter membrane is 100 nm.

[0014] According to a preferred embodiment, in step (6), the pre-freezing temperature is -60~-80℃, and the vacuum degree of freeze drying is less than 20 Pa.

[0015] One of the objectives of this invention is to provide the use of the above-described method for rapidly separating ginsenoside Rd from total ginsenosides in the preparation of ginsenoside Rd products.

[0016] One of the objectives of this invention is to provide the use of zein for the rapid separation of ginsenoside Rd from total ginsenosides.

[0017] The beneficial effects of this invention are: This invention discovers that by precisely controlling the self-assembly conditions (ethanol concentration, Zein / total saponin ratio, water ratio, etc.), zein can selectively encapsulate only Rd from a ginseng total saponin mixture during antisolvent precipitation by utilizing the difference in affinity between different saponin monomers and Zein, while other saponins (Rb1, Rg1, Re, etc.) are essentially not encapsulated. Therefore, this invention uses zein nanoparticles as a solid-phase extraction carrier, leveraging the selectivity difference between zein and ginseng saponin Rd to achieve highly selective recognition and enrichment of Rd during antisolvent self-assembly. After co-dissolving ginseng total saponins and zein, Rd separation and nano-encapsulation are simultaneously achieved through a single antisolvent precipitation. Compared to the traditional two-step method of first chromatographically purifying monomers and then encapsulating them separately, this method greatly simplifies the operation process, reducing operation time by approximately 80% and the separation cycle from several days to weeks in traditional methods to approximately 8 hours, making it suitable for large-scale production.

[0018] Meanwhile, unencapsulated free saponins can be removed by simple filtration using a filter membrane with a pore size of 50~200 nm. The freeze-dried nanoparticles can be extracted with a small amount of acetonitrile to obtain Rd monomers with a purity of ≥95%. The amount of organic solvent used is reduced by no less than 90%, mainly using inexpensive and readily available ethanol and water. This avoids the problems of traditional methods that use large amounts of expensive organic solvents such as acetonitrile and methanol, as well as the high cost and limited service life of chromatographic packing materials. The yield from total ginsenosides to Rd monomers is significantly higher than that of traditional preparative liquid chromatography, and the production cost is greatly reduced.

[0019] Furthermore, by precisely controlling the antisolvent self-assembly conditions, including key process parameters such as ethanol volume fraction of 60%~80%, zein to ginsenoside mass ratio of 0.5:1~2:1, and mixture to water volume ratio of 1:3~1:6, zein nanoparticles can selectively encapsulate only Rd from the complex saponin mixture, while other saponins are basically not encapsulated. The saponin components in the resulting extract are almost entirely ginsenoside Rd, and the residual amount of other saponins is negligible. Attached Figure Description

[0020] Figure 1 Liquid phase diagrams of mixed standard, total ginsenoside raw material, and purified ginsenoside extract under the same liquid phase conditions for liquid phase detection of ginsenoside Rd content after extraction using corn protein loading method; Figure 2 The yields of ginsenoside Rd prepared by liquid phase method and corn protein method; Figure 3 A transmission electron microscope image of corn protein nanoparticles loaded with ginsenoside Rd; Figure 4 Infrared spectra of corn protein nanoparticles with and without ginsenoside Rd; Figure 5 DSC spectra of corn protein nanoparticles with and without ginsenoside Rd encapsulation; Figure 6 To compare the loading rates of total ginsenosides and ginsenoside Rd in corn protein particles, total ginsenosides were determined by the sulfuric acid-phenol method, while ginsenoside Rd was determined by high performance liquid chromatography. Detailed Implementation

[0021] In the description of this invention, terminology is used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of indicated technical features. In the description of this invention, "a plurality of" means two or more, unless otherwise explicitly defined.

[0022] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods; materials, reagents, or instruments whose manufacturers are not specified are all commercially available; conditions not specified in the examples are performed according to conventional conditions or conditions recommended by the manufacturer. Furthermore, this invention does not limit the source of the raw materials used; unless otherwise specified, all raw materials used in this invention are commercially available products commonly found in this technical field. Zein was purchased from Beijing Solarbio Science & Technology Co., Ltd., with a purity ≥95%. Total ginsenosides were purchased from Shaanxi Jinkangtai Biotechnology Co., Ltd. Unless otherwise specified, all solvents used were of analytical grade. Polycarbonate filter membranes were purchased from Tianjin Jinteng Experimental Equipment Co., Ltd.

[0023] Example 1: Preparation of corn protein nanoparticles specifically encapsulating ginsenoside Rd and extraction of pure Rd product This embodiment provides a method for preparing corn protein nanoparticles that selectively encapsulate ginsenoside Rd directly from total ginsenosides, and further extracts high-purity ginsenoside Rd monomers from these nanoparticles. The specific steps are as follows: Step 1: Preparation of zein solution Weigh 1.0 g of zein (purity ≥95%) and dissolve it in 100 mL of 70% (v / v) ethanol-water solution. Stir magnetically at room temperature until completely dissolved to obtain a clear zein solution with a mass concentration of 1.0% (w / v).

[0024] Step 2: Dissolving total ginsenosides Add 1.0 g of ginsenoside solids (with an Rd content of 5% in total ginsenosides) to the above zein solution, and continue stirring until completely dissolved to obtain a mixture. The mass ratio of zein to ginsenosides in the mixture is 1:1.

[0025] Step 3: Preparation of nanoparticle solid dispersion by antisolvent precipitation At room temperature, the above mixture was slowly injected into 4 times its volume (400 mL) of distilled water at a volume ratio of 1:4, while maintaining magnetic stirring (800 rpm) during the injection process. After injection, stirring was continued for 30 min. The mixture changed from clear to a milky white suspension, thus forming a nanoparticle solid dispersion. During this process, zein self-assembled into nanoparticles due to a sudden decrease in polarity, while selectively encapsulating ginsenoside Rd.

[0026] Step 4: Remove ethanol The obtained nanoparticle solid dispersion was transferred to a rotary evaporator and evaporated under a water bath at 46°C and a vacuum of 0.08 MPa until no ethanol odor was detected (30 min), yielding a nanoparticle dispersion free of ethanol. This operation avoids the influence of residual ethanol on subsequent purification and freeze-drying processes.

[0027] Step 5: Remove unencapsulated ginsenosides The nanoparticle dispersion after ethanol removal was filtered using a polycarbonate membrane with a pore size of 100 nm. Unencapsulated free ginsenosides (including unencapsulated Rd and other saponins) could pass through the membrane due to their small molecular size; however, Rd-encapsulated corn protein nanoparticles (100–200 nm in diameter) were retained on the membrane. The retained nanoparticles were collected and washed twice with a small amount of distilled water to obtain a purified dispersion of corn protein nanoparticles specifically encapsulating ginsenoside Rd.

[0028] Step 6: Freeze-drying The nanoparticle dispersion obtained in step 5 was pre-frozen at -80℃ for 12 h, and then freeze-dried in a freeze dryer (vacuum degree <10 Pa, cold trap temperature -50℃) for 48 h to obtain corn protein freeze-dried particles specifically encapsulating ginsenoside Rd.

[0029] Step 7: Acetonitrile extraction to obtain pure ginsenoside Rd Weigh 100 mg of the lyophilized granules obtained in step 6, add 2 mL of acetonitrile, and extract by sonication for 10 min. Centrifuge (12000 rpm, 5 min) and collect the supernatant. Repeat the extraction twice and combine the supernatants. Evaporate the supernatant to dryness at 40℃ to obtain a white powdery solid, which is pure ginsenoside Rd. High-performance liquid chromatography (HPLC) analysis showed that the purity of Rd in this extract was ≥95%.

[0030] Comparative Example 1: Yield comparison with preparative liquid phase method Using the same mass (1.0 g) of total ginsenosides as raw materials, ginsenoside Rd was separated and purified by the method of the present invention (steps 1-7 of Example 1 above) and the traditional preparative high performance liquid chromatography (using a C18 column, acetonitrile-water gradient elution). The mass of the obtained pure Rd was calculated, and the yield (mass of pure Rd / mass of total ginsenosides in raw materials × 100%) was compared.

[0031] The results are as follows Figure 2 As shown, the Rd yield of the method of the present invention is significantly higher than that of the preparative liquid phase method (p<0.01), while the operation time is shortened by nearly 80% and the amount of organic solvent used is reduced by nearly 90%.

[0032] Example 1: Analysis of saponin composition in extracts by liquid chromatography Rd extract was prepared according to steps 1-7 of Example 1. Its saponin composition was analyzed by high-performance liquid chromatography (HPLC). A mixed saponin standard (containing Rd, Rb1, Rg1, and Re) and untreated ginseng total saponins were used as controls. Chromatographic conditions: C18 column (4.6 × 250 mm, 5 μm), acetonitrile-water gradient elution, detection wavelength 203 nm.

[0033] The results are as follows Figure 1 As shown, multiple saponin chromatographic peaks (including Rd, Rb1, Rg1, Re, etc.) are visible in the ginseng total saponin raw material; however, after extraction using the method of this invention, only a single main peak appears in the liquid chromatogram of the extract at the retention time corresponding to the Rd standard, while other saponin peaks essentially disappear. This indicates that corn protein nanoparticles achieve highly selective encapsulation and purification of ginseng saponin Rd, effectively removing other types of ginseng saponins.

[0034] Example 2: Observation of nanoparticle morphology using transmission electron microscopy Take the corn protein nanoparticle dispersion specifically embedded with Rd obtained in step 5, drop it onto a carbon film copper grid, negatively stain with 2% phosphotungstic acid, dry at room temperature, and observe under a transmission electron microscope (TEM, accelerating voltage 120 kV).

[0035] The results are as follows Figure 3 As shown, the nanoparticles are regularly spherical with a uniform particle size distribution, averaging 120–160 nm, and no obvious aggregation was observed. The nanoscale particle size indicates that the corn protein nanoparticles have a large specific surface area, which is beneficial for efficient Rd encapsulation and subsequent extraction.

[0036] Example 3: Infrared spectroscopy analysis of embedding interactions Unencapsulated blank zein nanoparticles (prepared in the same way as in Example 1 but without the addition of total ginsenosides) and zein nanoparticles encapsulated with Rd obtained in step 5 of Example 1 were respectively subjected to Fourier transform infrared spectroscopy (FTIR) scanning (4000~400 cm⁻¹). -1 ).

[0037] The results are as follows Figure 4 As shown: Compared with blank Zein nanoparticles, Rd-encapsulated nanoparticles show higher growth rates at 1650 cm⁻¹. -1 (Amide I band) and 1540 cm -1 Characteristic peak shifts and intensity changes occur at the (amide II band), and simultaneously at 1070 cm⁻¹ -1 The appearance of COC stretching vibration peaks attributable to Rd glycosidic bonds indicates that hydrogen bonds and hydrophobic interactions have formed between Rd and corn protein, confirming that Rd is effectively embedded inside the nanoparticles, rather than being a simple physical mixture.

[0038] Example 4: Differential Scanning Calorimetry for Thermal Stability Analysis The thermal denaturation temperatures of blank Zein nanoparticles and Rd-embedded Zein nanoparticles (the sample obtained in step 5) were determined using differential scanning calorimetry (DSC). 5 mg of sample was weighed and heated from 20 °C to 200 °C at a heating rate of 10 °C / min, and the heat flow curves were recorded.

[0039] The results are as follows Figure 5 As shown, the denaturation temperature of the blank Zein nanoparticles was 89.2℃; after Rd encapsulation, the denaturation temperature of the Zein nanoparticles increased to 96.5℃, and the enthalpy also increased significantly. This suggests that the introduction of ginsenoside Rd enhances the stability of the higher-order folded structure inside the corn protein particles, further confirming the successful encapsulation of Rd and the strong interaction between the two.

[0040] Example 5: Comparative Analysis of Total Saponin Content and Rd Content The nanoparticles obtained in step 5 of Example 1 were extracted with acetonitrile using ultrasonic extraction (same as step 7) to obtain an extract. The content of saponins in the extract was determined by two methods: (1) the total saponin content was determined by the sulfuric acid-phenol method (Rd was used as a reference, and the absorbance was measured at 490 nm after color development); (2) the content of ginsenoside Rd was determined by high performance liquid chromatography (HPLC) (chromatographic conditions were the same as in Example 1).

[0041] The results are as follows Figure 6 As shown, there was no significant difference between the total saponin content determined by the sulfuric acid-phenol method and the Rd content determined by HPLC (p>0.05), indicating that after selective encapsulation with corn protein nanoparticles, almost all the saponin components in the extract were ginsenoside Rd, and the residual amounts of other saponins were negligible. This result is consistent with... Figure 1 The results of liquid chromatography corroborate each other, fully demonstrating that this method has excellent selective embedding effect.

[0042] In summary, the method for preparing corn protein nanoparticles specifically encapsulating ginsenoside Rd provided by this invention can directly use inexpensive total ginsenosides as raw materials. Through the self-assembly process of corn protein nanoparticles, highly selective enrichment and encapsulation of ginsenoside Rd are achieved, and high-purity Rd monomers can be easily extracted. Compared with traditional liquid-phase methods, this invention significantly simplifies the operation process, reduces organic solvent consumption and preparation costs, while achieving a higher yield. Figure 2 This provides a novel technical solution for the industrial production of ginsenoside Rd and its nano-formulations.

[0043] It should be noted that the specific embodiments described above are exemplary, and those skilled in the art can devise various solutions inspired by the disclosure of this invention, all of which fall within the scope of this invention and its protection. Those skilled in the art should understand that this specification is illustrative and not intended to limit the scope of the claims. The scope of protection of this invention is defined by the claims and their equivalents.

Claims

1. A method for rapidly separating ginsenoside Rd from total ginsenosides, characterized in that, Includes the following steps: (1) Dissolve zein in an ethanol solution to obtain a zein solution; (2) Dissolve the total ginsenosides in the zein solution to obtain a mixture; (3) The mixture is slowly injected into water, and under stirring conditions, zein is triggered to self-assemble into nanoparticles, while ginsenoside Rd is selectively encapsulated to obtain a nanoparticle dispersion. (4) The ethanol in the nanoparticle dispersion is removed by rotary evaporation; (5) The nanoparticle dispersion is filtered with a filter membrane with a pore size smaller than that of the nanoparticles to retain the zein nanoparticles containing ginsenoside Rd, wherein the nanoparticles have a particle size of 100~200 nm, and the unencapsulated free ginsenosides are removed to obtain a purified nanoparticle dispersion. (6) The purified nanoparticle dispersion was pre-frozen and then freeze-dried to obtain zein freeze-dried particles containing ginsenoside Rd. (7) The freeze-dried particles were ultrasonically extracted with acetonitrile, and the supernatant was collected after centrifugation to obtain ginsenoside Rd with a purity ≥95%.

2. The method according to claim 1, characterized in that, In step (1), the volume fraction of the ethanol solution is 60%~80%.

3. The method according to claim 2, characterized in that, In step (1), the volume fraction of the ethanol solution is 70%.

4. The method according to claim 1, characterized in that, In step (2), the mass ratio of zein to total ginsenosides is 0.5 to 2:

1.

5. The method according to claim 4, characterized in that, In step (2) and step (3), the volume ratio of the mixture to water is 1:3~6.

6. The method according to claim 1, characterized in that, In step (4), the temperature of rotary evaporation is 40~50℃.

7. The method according to claim 1, characterized in that, In step (5), the pore size of the filter membrane is 100 nm.

8. The method according to claim 1, characterized in that, In step (6), the pre-freezing temperature is -60~-80℃, and the vacuum degree of freeze drying is less than 20 Pa.

9. The method for rapidly separating ginsenoside Rd from total ginsenosides according to any one of claims 1-9 is used in the preparation of ginsenoside Rd products.

10. Application of zein in the rapid separation of ginsenoside Rd from total ginsenosides.

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