Process for extracting gypenoside C from gypenoside leaves

Abstract

The application discloses a process for extracting gypenoside C from Paeonia lactiflora Pall. leaves and relates to the technical field of natural product separation and purification. The process comprises the following steps: taking the Paeonia lactiflora Pall. leaves as raw materials, adopting ethanol aqueous solution reflux extraction and concentration drying to obtain an extract; dissolving the extract in ethanol aqueous solution, adding a flocculating agent, adjusting pH, performing solid-liquid separation, and collecting flocculated liquid; loading the flocculated liquid onto a macroporous resin column, performing gradient elution enrichment, and obtaining macroporous resin enrichment liquid; further purifying the macroporous resin enrichment liquid through a medium-pressure ODS column gradient elution to obtain a medium-pressure purified product; and finally performing isocratic elution through high-pressure preparative liquid chromatography, and collecting a target flow fraction. The process can efficiently remove impurities such as cellulose, pectin and pigment in the leaves, does not need superhigh pressure or special customized equipment, is simple in steps, easy to control in parameters, can stably obtain gypenoside C products with a purity higher than 98%, realizes comprehensive utilization of Paeonia lactiflora Pall. plant resources, and is suitable for large-scale production.

Description

Technical Field

[0001] This invention relates to the field of natural product separation and purification technology. More specifically, this invention relates to a process for extracting hederacin C from the leaves of Pulsatilla chinensis. Background Technology

[0002] White-headed old man ( Pulsatillachinensis Pulsatilla chinensis is a plant belonging to the genus Pulsatilla in the family Ranunculaceae. Its dried root is a traditional Chinese medicine with the effects of clearing heat and cooling blood, detoxifying and stopping dysentery. It is widely used in the treatment of diseases such as dysentery caused by heat toxins and blood stasis, and carbuncles and boils. Hederagenin C (HSC) is the core triterpenoid saponin active ingredient in Pulsatilla chinensis, CAS number: 14216-03-6. It has multiple potential biological activities, including strong anti-inflammatory effects, cartilage protection, benefits to the respiratory system, antibacterial and analgesic effects. It exerts its anti-inflammatory effect by inhibiting the key inflammatory pathways of NF-κB and MAPK, providing an important material basis for the development of related anti-inflammatory drugs.

[0003] Currently, research on the extraction of ivy saponin C focuses on the roots of Pulsatilla chinensis. Existing techniques include various methods for extracting and purifying saponins from Pulsatilla chinensis roots, but all use the roots as raw materials. Research on Pulsatilla chinensis leaves is limited to qualitative analysis and content determination of chemical components; no reports have been found on the process of extracting and preparing ivy saponin C from Pulsatilla chinensis leaves.

[0004] Through systematic research, the inventors discovered that the content of ivy saponin C in Pulsatilla chinensis leaves (2.1%) is significantly higher than that in the roots (0.4%), approximately five times that of the roots. However, Pulsatilla chinensis leaves contain a large amount of impurities such as cellulose, pectin, tannins, and pigments. These impurities are easily co-extracted with the target component, interfering with the separation and purification process and contaminating the purification packing material. This results in low purification efficiency and difficulty in achieving the required product purity when directly applying existing root extraction processes. Therefore, developing an extraction and purification process specifically for Pulsatilla chinensis leaves that can efficiently remove impurities and stably obtain high-purity ivy saponin C is a pressing technical problem to be solved in this field and represents the technological breakthrough that this invention aims to achieve. Summary of the Invention

[0005] This invention provides a process for extracting hederone C from Pulsatilla chinensis leaves. It is the first time that Pulsatilla chinensis leaves have been used as raw material, and a unique extraction process specifically designed for Pulsatilla chinensis leaves has been proposed. While ensuring a high HSC recovery rate (≥96%), it can stably obtain hederone C with a purity of >98%. The process is simple, the parameters are easy to control, and it avoids the problems of complex operation and high cost. It balances efficiency and practicality and can be fully put into actual production, providing support for the large-scale acquisition of hederone C.

[0006] To achieve these objectives and other advantages according to the present invention, a process for extracting hederacin C from the leaves of Pulsatilla chinensis is provided, comprising the following steps: S1. Extraction steps: The leaves of Pulsatilla chinensis were extracted by reflux with an ethanol-water solution. The extract was then concentrated and dried to obtain crude extract of Pulsatilla chinensis leaves. S2, Flocculation and Impurity Removal Step: Dissolve the crude extract of Pulsatilla chinensis leaves in an ethanol aqueous solution, add flocculant for flocculation treatment and adjust the pH to 7-8, and obtain the flocculated liquid after solid-liquid separation; S3. Macroporous resin enrichment step: The flocculated liquid is loaded onto a macroporous resin column and eluted with ethanol-water solution as the mobile phase. The eluted portion rich in ivy saponin C is collected to obtain the macroporous resin enrichment solution. S4. Medium-pressure chromatographic purification step: Load the macroporous resin enrichment solution onto a reversed-phase silica gel preparative chromatographic column, elute with methanol-water solution as the mobile phase, collect the target fraction, and obtain the medium-pressure purified product. S5. High-pressure preparation and purification step: The medium-pressure purified product is loaded onto a preparative high-performance liquid chromatography column, eluted with acetonitrile-water solution, and the fraction corresponding to the target chromatographic peak is collected to obtain high-purity ivy saponin C.

[0007] Preferably, in step S1, the leaves of Pulsatilla chinensis are refluxed and extracted 1-3 times with an ethanol aqueous solution with a volume concentration of 70% to 80%. The extracts are combined, concentrated and dried to obtain an extract, which is then dissolved in an ethanol aqueous solution with a volume concentration of 15% to 25%. After standing and separating into layers, the supernatant is collected, concentrated and dried again to obtain a crude extract of Pulsatilla chinensis leaves.

[0008] Preferably, in step S2, the flocculant is polyaluminum chloride. The crude extract of Pulsatilla chinensis leaves is dissolved in an aqueous ethanol solution with a volume concentration of 15% to 25%, polyaluminum chloride is added for flocculation treatment and the pH is adjusted to 7-8. After solid-liquid separation, the precipitate obtained by separation is washed with an aqueous ethanol solution with a volume concentration of 15% to 25%, and the washing liquid is combined with the liquid obtained by solid-liquid separation to obtain the flocculated liquid.

[0009] Preferably, in step S2, the flocculant is polyaluminum chloride, and the flocculation treatment employs staged pH-controlled circulating flocculation, specifically including: S21. The crude extract of Pulsatilla chinensis leaves dissolved in an ethanol aqueous solution with a volume concentration of 15% to 25% was first adjusted to pH 4 to 5 with acid, polyaluminum chloride was added, and after stirring and reacting, the pH was adjusted to 7-8 in stages with alkaline solution. After standing, aluminum hydroxide precipitate was fully generated, and solid-liquid separation was performed to obtain the first-stage flocculated liquid and the first-stage flocculated precipitate. S22. After washing the first-stage flocculation precipitate with an ethanol aqueous solution with a volume concentration of 15% to 25%, it is resuspended in an ethanol aqueous solution with a volume concentration of 15% to 25%. The pH is adjusted to 4 to 5 with citric acid, and the aluminum hydroxide precipitate is dissolved by stirring. After standing, the solid and liquid are separated to obtain the acid treatment solution. S23. Adjust the pH of the acid treatment solution to 7-8 with alkaline solution, let it stand to allow aluminum hydroxide precipitate to be fully formed, and then separate the solid and liquid after standing to obtain the second-stage flocculated liquid. S24. Combine the first-stage flocculated liquid with the second-stage flocculated liquid to obtain the flocculated liquid.

[0010] Preferably, the elution in step S3 is gradient elution, first eluting 3 to 5 column volumes with an ethanol-water solution of 15% to 25% (v / v), and then eluting 4 to 6 column volumes with an ethanol-water solution of 45% to 55% (v / v).

[0011] Preferably, in step S3, the macroporous resin column is pretreated before use, the pretreatment including soaking in ethanol and washing with water until there is no alcohol odor.

[0012] Preferably, the macroporous resin in step S3 is one of type D101, type LX-20B, and type AB-8.

[0013] Preferably, the elution in step S4 is gradient elution, first eluting with a methanol-water solution with a volume concentration of 8% to 12% for 0.5 to 1.5 column volumes, and then gradually increasing the methanol concentration to 55% to 65% for 5 to 7 column volumes.

[0014] Preferably, the elution in step S5 is isocratic elution, using an acetonitrile-water solution with a volume concentration of 25% to 30%.

[0015] Preferably, in step S5, the medium-pressure purified product is concentrated to obtain a solid, which is then dissolved in methanol and loaded onto a preparative high-performance liquid chromatography column.

[0016] The present invention has at least the following beneficial effects: First, this invention is the first to extract ivy saponin C from Pulsatilla chinensis leaves, opening up a completely new source of raw materials. In existing technologies, the extraction and purification of ivy saponin C have all used roots as raw materials, with no reports of using leaves. This invention, for the first time, successfully prepared high-purity ivy saponin C using Pulsatilla chinensis leaves as raw materials, providing a new and sustainable source of this highly active natural product. It avoids the damage to medicinal plant resources caused by traditional root harvesting, and has significant ecological benefits.

[0017] Secondly, this invention is the first to reveal and utilize the characteristic that the HSC content in Pulsatilla chinensis leaves is higher than that in the roots. Through systematic content determination, the inventors found that the HSC content in Pulsatilla chinensis leaves (2.1%) is about 5 times that in the roots (0.4%). This invention fully utilizes this characteristic, using leaves as raw materials to enhance the enrichment potential of the target component from the source, laying a material foundation for the efficient operation of subsequent purification processes.

[0018] Third, this invention establishes a dedicated purification process for the impurity profile of Pulsatilla chinensis leaves. Pulsatilla chinensis leaves contain a large amount of impurities such as cellulose, pectin, tannins, and pigments. Directly applying existing root purification processes can lead to resin contamination, column clogging, and substandard product purity. This invention, targeting the characteristics of the leaf impurity profile, introduces a flocculation and impurity removal step at the front end of the purification process for the first time. This effectively removes large molecular impurities such as pigments, achieving front-end impurity removal and back-end load reduction. Combined with gradient elution with macroporous resin and multi-stage chromatographic purification, a complete, stable, and efficient leaf-specific purification process is formed.

[0019] Fourth, in the flocculation and impurity removal step, this invention employs graded pH-controlled cyclic flocculation. The first adjustment of the pH to 7-8 converts aluminum ions in polyaluminum chloride into aluminum hydroxide precipitate, which is then separated and removed, reducing the contamination of aluminum ions in the solution by subsequent purification units. The precipitate is then acid-treated to dissolve the aluminum hydroxide and release the encapsulated ivy saponin C. The pH of the acid-treated solution is then adjusted back to 7-8 to allow the aluminum ions to reprecipitate and separate. No new polyaluminum chloride is added in this step, achieving a significant increase in HSC recovery rate while efficiently removing aluminum ions.

[0020] Fifth, the process of this invention is stable and reproducible. While achieving a high HSC recovery rate (flocculation step ≥96%), it can stably obtain a high-purity product with a purity >98%. The process of this invention has been verified through multiple batches and can stably obtain ivy saponin C with a purity greater than 98%, fully meeting the quality requirements of chemical reference standards and high-purity reagents. This provides technical support for the large-scale preparation and in-depth pharmacological research of ivy saponin C.

[0021] Other advantages, objectives and features of the present invention will become apparent in part from the following description, and in part from those skilled in the art through study and practice of the invention. Attached Figure Description

[0022] Figure 1 The standard curve of HSC for one technical solution of the present invention; Figure 2 This is a comparison diagram of the root and leaves of Pulsatilla chinensis and HSC standard samples according to one technical solution of the present invention; Figure 3 This is a chromatographic comparison of samples before and after the addition of flocculant according to one technical solution of the present invention; Figure 4 This is a liquid chromatogram of the washing liquid after centrifugation and precipitation following the addition of flocculant, according to one technical solution of the present invention. Figure 5 This is a liquid chromatogram of macroporous resin after coarse separation according to a technical solution of the present invention; Figure 6 This is a real-time monitoring diagram of the medium-pressure ODS column purification process according to one technical solution of the present invention; Figure 7 The liquid chromatogram of the combined fractions after medium-pressure preparation according to a technical solution of the present invention; Figure 8 This is a real-time elution monitoring graph of high-pressure preparative liquid chromatography according to one technical solution of the present invention; Figure 9 This is an HPLC purity detection chromatogram of the final product of Example 9 of the present invention; Figure 10 This is an HPLC purity test result of the final product of Example 10 of the present invention. Detailed Implementation

[0023] The present invention will now be described in further detail with reference to the accompanying drawings, so that those skilled in the art can implement it based on the description.

[0024] It should be understood that terms such as “having,” “comprising,” and “including” as used herein do not exclude the presence or addition of one or more other elements or combinations thereof.

[0025] It should be noted that, unless otherwise specified, the test methods described in the following embodiments are all conventional methods, and the reagents and materials described are all commercially available unless otherwise specified, and therefore should not be construed as limiting the present invention.

[0026] It is worth noting that this invention is the first to extract hedyotis diffusa saponin C from the leaves of *Pulsatilla chinensis*. In the prior art, research on the extraction of hedyotis diffusa saponin C has focused on the roots, with no reports of processes using leaves as raw materials. The inventors discovered that the HSC content in *Pulsatilla chinensis* leaves (2.1%) is significantly higher than that in the roots (0.4%), approximately five times that of the roots. However, the content of impurities (cellulose, pectin, tannins, pigments, etc.) in the leaves is also much higher than in the roots, making it difficult to obtain a high-purity product by directly applying existing root purification processes. Therefore, this invention, targeting the characteristics of leaf impurity profiles, developed a proprietary process of flocculation-macroporous resin enrichment-medium-pressure chromatographic purification-high-pressure final purification. The core objective of the flocculation step is to remove aluminum ions introduced by polyaluminum chloride, protecting the adsorption sites of the subsequent macroporous resin and the chromatographic packing material, while simultaneously improving HSC recovery. The core objective of the chromatographic step is to improve HSC purity. The synergistic effect of these two steps achieves a balance between high recovery (≥94%) and high purity (>98%), successfully realizing the efficient and stable preparation of high-purity hederone C from Pulsatilla chinensis leaves. In the flocculation step, this invention innovatively employs a graded pH-controlled cyclic flocculation method, through two alkali adjustment-precipitation-separation operations (without adding new polyaluminum chloride in the second operation), reducing the residual aluminum ion concentration in the flocculated solution from 118.6 mg / L in conventional processes to 3.2 mg / L. mg / L (removal rate 97.3%), thus effectively avoiding aluminum ions occupying the adsorption sites of macroporous resin and preventing them from contaminating the chromatographic packing. Acid treatment releases HSCs encapsulated by aluminum hydroxide precipitate, increasing the HSC recovery rate from 72.0% of conventional one-time flocculation to 94.8%, achieving the dual goals of aluminum removal to protect the packing and high recovery rate.

[0027] <Establishment of a method for determining the content of ivy saponin C> The purpose of this experiment is to establish a quantitative analysis method for hederaside C (hereinafter referred to as HSC) and to compare its content differences in the roots and leaves of Pulsatilla chinensis.

[0028] 1.1 Establishment of High Performance Liquid Chromatography (HPLC) Analysis Method The chromatographic conditions were as follows: acetonitrile-water (26:74 v / v) as the mobile phase, a C18 column (4.6 mm × 250 mm, 5 μm), a flow rate of 1.0 mL / min, a detection wavelength of 210 nm, and an injection volume of 20 μL.

[0029] Accurately weigh the HSC reference standard, dissolve and dilute it in methanol to prepare a series of solutions with known concentrations, and analyze them under the chromatographic conditions described above. Perform linear regression with the reference standard concentration as the x-axis (X, μg) and the peak area as the y-axis. The standard curve for ivy saponin C is shown below. Figure 1 As shown, the equation of the standard curve is Y = 5.89 × 10⁻⁶. 6 X-1.46×10 4Correlation coefficient R 2 =0.9992. The results show that HSC exhibits good linearity in the range of 2–32 μg.

[0030] The method was validated for precision, stability, recovery, and repeatability. The relative standard deviations (RSD) of instrument precision, sample solution stability, and method repeatability were all less than 3.0%, and the recovery rates were between 95% and 102%, indicating that the method is accurate and reliable and can be used for content determination.

[0031] 1.2 Prepare test solutions of Pulsatilla root and leaves and measure their content. Accurately weigh 0.1 g each of Pulsatilla root powder and Pulsatilla leaf powder, place them in a stoppered conical flask, accurately add 25 mL of 75% methanol, weigh the flask, sonicate for 30 min, cool, replenish the lost weight, shake well, filter, and take the filtrate as the test solution.

[0032] Root and leaf test solutions of Pulsatilla chinensis were prepared. These solutions, along with the HSC reference solution, were injected separately into a liquid chromatograph and analyzed under the aforementioned chromatographic conditions. Qualitative analysis was performed by comparing retention times, and the content was calculated based on the standard curve.

[0033] A comparison chart of Pulsatilla root extract, Pulsatilla leaf extract, and pure hederaside C is shown below. Figure 2 As shown in the figure, the horizontal axis represents retention time (t / min), and the vertical axis represents the detection response value. A comparison of the liquid chromatography results of Pulsatilla chinensis leaf extract (blue curve), Pulsatilla chinensis root extract (red curve), and pure hederaside C (black curve) shows that the HSC content in Pulsatilla chinensis root extract is approximately 0.4%, while the HSC content in Pulsatilla chinensis leaf extract is approximately 2.1%. This result confirms that the HSC content in Pulsatilla chinensis leaf extract is significantly higher than that in Pulsatilla chinensis root extract, approximately five times that of the root, indicating that Pulsatilla chinensis leaves are a potentially high-quality raw material for obtaining this active ingredient, but the leaf tip contains more impurities. The peak position of the black curve represents the retention time of the HSC-specific characteristic peak, which is the core basis for determining whether other samples contain HSC.

[0034] <Optimization of Flocculation and Impurity Removal Process and Its Impact on Purification> This experiment addresses the issue of numerous impurities in Pulsatilla leaf extract by investigating the effect of polyaluminum chloride (PAC) flocculant dosage on impurity removal efficiency and HSC recovery rate.

[0035] 2.1 Experimental Methods and Results Approximately 1 g of concentrated and dried Pulsatilla chinensis leaf crude extract powder was accurately weighed and dissolved in 20 mL of 20% ethanol. Six experimental groups were set up, with 0, 1, 2, 3, 4, and 5 mL of 10% polyaluminum chloride solution added respectively. The mixture was placed in a shaker at room temperature and shaken overnight (120 rpm) to ensure sufficient flocculation reaction. The pH was adjusted to 7-8 with saturated sodium carbonate. The supernatant was collected by centrifugation and brought to a final volume of 50 mL. The HSC content was determined, and the recovery rate was calculated. The flocculation and impurity removal effect is shown in Table 1.

[0036] Table 1 As shown in Table 1, the color of the sample solution became significantly lighter as the amount of polyaluminum chloride increased from 0 mL to 5 mL, clearly indicating that pigment impurities were effectively flocculated and precipitated. With increasing flocculant dosage, the recovery rate of HSC in the supernatant gradually decreased. When 5 mL of flocculant was added, the recovery rate of HSC in the supernatant was 72.0%, indicating that while the flocculation process removes impurities, it also leads to the loss of some target products due to co-precipitation.

[0037] The precipitate formed after adding 5 mL of flocculant was washed with 20% ethanol solution, and the washing solution was measured. The precipitate recovery rate is shown in Table 2.

[0038] Table 2 As shown in Table 2, approximately 11.07 mg of HSC can be recovered from the precipitate. Combined with 65.24 mg in the supernatant, the total recovery reaches 76.31 mg, and the total recovery rate increases to 84.3%. This indicates that washing the precipitate can effectively reduce the loss of HSC caused by flocculation. Adding an appropriate amount of flocculant (5 mL) and combining it with the precipitate washing step can achieve effective front-end impurity removal while maintaining a high total HSC recovery rate.

[0039] <Effect Verification of Graded pH-Controlled Cyclic Flocculation Method> The purpose of this experiment is to verify the effectiveness of the graded pH-controlled cyclic flocculation method for aluminum removal and protection of the packing material.

[0040] 4.1 Experimental Methods and Results Each sample of concentrated and dried Pulsatilla chinensis leaf crude extract powder was accurately weighed and dissolved in 20% ethanol to a final volume of 50 mL. The following experimental groups were set up.

[0041] The control group used conventional one-time flocculation: 5 mL of 10% polyaluminum chloride was added, the pH was adjusted to 7-8 with saturated sodium carbonate, the mixture was stirred for 2 hours, and after standing for 2 hours, the solid and liquid were separated, and the supernatant was taken as the flocculated liquid.

[0042] The experimental group adopted a staged pH-controlled cyclic flocculation method: first, the pH was adjusted to 4-5, polyaluminum chloride was added, and after stirring and reaction, the pH was adjusted to 7-8 in stages with alkaline solution. After standing, solid-liquid separation was performed to obtain the first-stage flocculated liquid and the first-stage flocculated precipitate. The first-stage flocculated precipitate was washed and resuspended in an ethanol aqueous solution, the pH was adjusted to 4-5, and after stirring, solid-liquid separation was performed to obtain the acid-treated liquid. The pH of the acid-treated liquid was adjusted to 7-8, and after standing, solid-liquid separation was performed to obtain the second-stage flocculated liquid. The first-stage flocculated liquid and the second-stage flocculated liquid were combined to obtain the flocculated liquid.

[0043] Determination of residual aluminum ions in flocculated solution: The residual aluminum ions in each group of flocculated solutions were determined by ICP-OES. 3+ concentration; Saturated adsorption capacity of macroporous resin: Take an equal volume of flocculated liquid (adjusted to the same HSC concentration of about 1.5 mg / mL), load it onto an AB-8 macroporous resin column (resin amount 10 g) at a rate of 1 BV / h, elute with 20% ethanol for 4 BV, desorb with 50% ethanol, collect the eluent, determine the total HSC by HPLC, and calculate the saturated adsorption capacity of the resin (mg HSC / g resin). HSC recovery rate: The total amount of HSC in the flocculated liquid was determined by HPLC and divided by the total amount of HSC in the crude extract to calculate the recovery rate. The experimental results are shown in Table 3.

[0044] Table 3 As can be seen from Table 3, the Al of the flocculated liquid 3+ Concentration: In the control group, polyaluminum chloride was added once and the pH was directly adjusted to 7-8. The Al in PAC... 3+ Most of the aluminum ions were converted into Al(OH)3 precipitate. However, due to incomplete precipitation (small colloidal particles, insufficient settling time, or the presence of some soluble aluminum complexes), a high concentration of aluminum ions remained in the supernatant. A staged pH-controlled cyclic flocculation method was adopted. The specific process was as follows: the first adjustment of alkali generated Al(OH)3 precipitate and separated it. Acid treatment was used to dissolve the precipitate. The second adjustment of alkali generated Al(OH)3 precipitate again and separated it. The two precipitation-separation operations made the aluminum ions almost completely removed, with the residual concentration as low as 3.2 mg / L.

[0045] As shown in Table 3, the saturated adsorption capacity of the macroporous resin is significantly reduced. In the control group, residual aluminum ions (118.6 mg / L) in the supernatant will compete with saponins for adsorption sites during subsequent macroporous resin adsorption. 3+It is possible that the aluminum ion residue occupies the polar or weakly polar sites of the resin through electrostatic interaction or coordination, resulting in a reduction of effective adsorption sites and thus reducing the saturated adsorption capacity of HSC. The experimental group had extremely low aluminum ion residue, and the resin adsorption sites were almost not occupied by aluminum ions. Therefore, it was able to fully exert its adsorption capacity for HSC. The low aluminum ion residue effectively protected the saponin adsorption sites of the macroporous resin.

[0046] As shown in Table 3, during the single-stage flocculation in the control group, Al(OH)3 precipitate formed rapidly, encapsulating a large amount of HSCs in the solution. After solid-liquid separation, the HSCs were lost with the precipitate, resulting in a low recovery rate. In the experimental group, the first stage of alkali adjustment caused impurities to precipitate in stages, reducing the dense encapsulation structure and lowering the initial encapsulation of HSCs. Acid treatment (pH 4-5) dissolved the Al(OH)3 precipitate, releasing the encapsulated HSCs back into the solution. The second stage of alkali adjustment further dissolved the Al(OH)3 precipitate. 3+ The HSCs are redeposited, while the HSCs remain in the solution, thus avoiding re-encapsulation.

[0047] Comparison of HSC adsorption and desorption performance of different types of macroporous resins The purpose of this experiment is to verify the adsorption and purification effect of macroporous resin on ivy saponin C.

[0048] 5.1 Resin Activation Soak appropriate amounts of each type of resin in anhydrous ethanol for 24 hours, then wash with purified water until no alcohol odor remains, and dry for later use. The sources of each type of resin are shown in Table 4.

[0049] Table 4 5.2 Static Adsorption Test The crude extract of Pulsatilla chinensis leaves was redissolved in 20% ethanol and allowed to stand. The supernatant was then used as the test solution. 1 g of each type of pretreated macroporous resin was weighed and placed in a stoppered conical flask. 100 mL of the test solution was added, and the flask was weighed. The mixture was shaken at 120 rpm and 37℃ for 24 h, and weighed again. After making up the weight loss, the sample was taken. The resin was filtered, 100 mL of 95% ethanol was added, and the mixture was weighed. After shaking at 120 rpm and 37℃ for 12 h, the mixture was weighed again. After making up the weight loss, the supernatant was taken to determine the content of ivy saponin C. The adsorption capacity, desorption rate, and specific adsorption capacity were calculated. The static adsorption results are shown in Table 5.

[0050] Table 5 An ideal purification resin should possess both high adsorption capacity and excellent desorption efficiency. Table 5 shows that different resins exhibit significant differences in HSC adsorption capacity, ranging from 50.51 mg to 126.01 mg. This difference mainly stems from variations in resin polarity and specific surface area, affecting the interaction force between the resin and HSC molecules. Higher adsorption capacity means that the resin can hold more target components per unit mass, which is fundamental for purification and enrichment. The desorption rate directly relates to the efficient recovery of the target components. The desorption rates of different resins range from 75.0% to 96.6%. A high desorption rate indicates that under the given elution conditions, HSC can effectively detach from the resin and enter the eluent, which is crucial for ensuring process yield. If the desorption rate is low, even with high adsorption capacity, a large amount of HSC remains in the resin and cannot be recovered, resulting in low purification efficiency. The AB-8 resin exhibited a good adsorption capacity (97.37 mg) and achieved the highest desorption rate (96.6%), indicating that its adsorption force on HSC is moderate, it is easy to elute, and it has high overall purification potential. Resins such as D101 and LX-20B also showed similar excellent characteristics, providing a key basis for the selection of resins in subsequent dynamic purification processes.

[0051] 5.3 Dynamic Adsorption Test Based on the static adsorption test results, macroporous resins D101, LX-20B, LX-69B, and AB-8, which have high adsorption capacity and desorption rate, were selected for dynamic adsorption tests to simulate a column chromatography process closer to actual production. The Pulsatilla chinensis leaf extract was redissolved in 20% ethanol and allowed to stand. The supernatant was then used as the test solution. 10g of the pretreated resin was weighed, packed into a column with pure water, and four column volumes of the test solution were loaded at 1 BV / h to ensure sufficient adsorption of HSCs onto the resin. Four column volumes were then eluted with 20% ethanol to remove some water-soluble impurities. Finally, 95% ethanol was used for elution. Each eluent was collected, precisely measured, and the HSC content was calculated. The adsorption capacity, desorption rate, and specific adsorption capacity were then calculated. The dynamic adsorption results are shown in Table 6.

[0052] Table 6 As shown in Table 6, during the continuous dynamic loading process, D101 and LX-20B resins exhibited the highest dynamic adsorption capacities (661.20 mg and 668.58 mg, respectively). This indicates that under dynamic flow conditions, these two resins can more effectively capture the flowing HSC molecules and have higher practical processing capacity. In the subsequent 95% ethanol elution step, the desorption rates of D101 and LX-20B resins both exceeded 99%, and AB-8 resin also reached 97.9%. The extremely high desorption rates prove that the elution conditions can almost completely recover the adsorbed HSCs into the eluent, ensuring the process yield. Collecting this portion of the eluent yields the macroporous resin enrichment solution.

[0053] The entire dynamic process (sample loading - low-ethanol elution - high-ethanol elution) constitutes a complete purification unit. Through this step, HSCs are selectively enriched from the complex, high-impurity flocculated solution into the macroporous resin enrichment solution. Although the impurity removal rate was not directly measured, the design of the low-concentration ethanol elution section and the changes in the physicochemical properties of the final enrichment solution (such as color lightening) indicate that impurities such as pigments were effectively removed. This step significantly increases the relative content of HSCs in the solution, laying the foundation for subsequent higher-precision chromatographic purification and effectively reducing the pressure on the downstream purification process.

[0054] <Example 1> The process for extracting ivy saponin C from the leaves of Pulsatilla chinensis includes the following steps: S1. Extraction steps: Weigh 1.5 kg of Pulsatilla chinensis leaves and add 14 times the volume of 75% ethanol aqueous solution to reflux extract the leaves three times: 1.5 h for the first time, 1 h for the second time, and 1 h for the third time. Combine the extracts, concentrate and dry to obtain the extract, dissolve in 20% ethanol aqueous solution, allow to stand and separate into layers, take the supernatant, concentrate and dry again to obtain solid powder. The crude extract of Pulsatilla chinensis leaves is 317.2 g. The HSC content in the raw material (Pulsatilla chinensis leaves) is 2.1% calculated by the two-point external standard method, and the HSC content in the powder (crude extract of Pulsatilla chinensis leaves) is 9%, with an extraction rate of 90.6%. S2. Flocculation and impurity removal step: Dissolve 100 g of crude extract of Pulsatilla chinensis leaves in 1 L of 20% (v / v) ethanol aqueous solution, add 500 mL of flocculant (10% (w / w) polyaluminum chloride aqueous solution) for flocculation treatment and adjust the pH to 7-8, perform solid-liquid separation, wash the separated precipitate twice with 20% ethanol aqueous solution, and combine the washing liquid with the liquid obtained from solid-liquid separation to obtain the flocculated liquid. Calculate the HSC content in the flocculated liquid using the external standard two-point method to be 7.5 g, with a recovery rate of 83.3% relative to the original sample. Chromatographic comparison of samples before and after adding flocculant is shown below. Figure 3As shown, the horizontal axis represents retention time, and the vertical axis represents the detection response value, representing the content / concentration signal of the component. The curve without flocculant shows a large number of strong response peaks in the early retention time region (corresponding to polar impurities). These peaks mask the signal at the HSC characteristic peak position, causing interference with the target analyte signal. After adding 5 mL of flocculant, the response of the early peaks in the curve is significantly reduced (impurities are removed by flocculation and precipitation), and the signal at the HSC characteristic peak position is clearly retained. This indicates that the flocculant removed impurities, enriched the target analyte HSC, and achieved the effect of purifying the target analyte. The flocculation and impurity removal step reduces interference from pigments and polar impurities (resulting in simpler peak shapes) while ensuring the retention of HSC, providing a feed solution with fewer impurities for subsequent macroporous resin enrichment and chromatographic purification.

[0055] The liquid chromatogram of the precipitate after centrifugation following the addition of 5 mL of polyaluminum chloride and washing with 20% ethanol is shown below. Figure 4 As shown, the horizontal axis represents retention time, and the vertical axis represents the detection response value, representing the content / concentration signal of the component. The presence of HSC characteristic peak signals in the figure indicates that the flocculated precipitate contains not only impurities but also some HSC. Precipitation washing can recover the HSC carried in the precipitate, avoiding the trace loss of the target substance and directly improving the total recovery rate of ivy saponin C. This achieves both impurity removal through flocculation and reduction of target substance loss through washing.

[0056] S3. Macroporous resin enrichment procedure: The macroporous resin column (LX-20B type) was soaked in ethanol and then washed with water until no alcohol odor remained. The flocculated solution was loaded onto the macroporous resin column at a flow rate of 1 BV / h. After loading, gradient elution was performed using ethanol-water solution as the mobile phase. First, 4 column volumes were eluted with ethanol-water solution of 15% to 25% v / h, and then 5 column volumes were eluted with ethanol-water solution of 50% v / h. The eluent rich in ivy saponin C was collected to obtain the macroporous resin enrichment solution. The macroporous resin content in the 50% elution fraction was calculated to be 1.3 mg / mL using the external standard two-point method, containing a total of 6.5 g of HSC. The recovery rate was 86.6% relative to the loaded sample amount. The liquid chromatogram after coarse separation by macroporous resin is shown below. Figure 5 As shown, the horizontal axis represents retention time, and the vertical axis represents the detection response value. The macroporous resin serves as a coarse enrichment agent. Gradient elution (from 20% to 50% ethanol) achieves preliminary separation of impurities from the target analyte. The liquid phase diagrams of the samples after flocculation and impurity removal are compared. Figure 3(The curve after adding flocculant) shows that the impurity peaks have basically disappeared and the target analyte response is stronger, indicating that the 20% ethanol elution step effectively removed most of the polar impurities. The proportion of the target analyte in the 50% ethanol eluent is significantly increased, and the characteristic peak response of the target analyte is stronger, proving that the macroporous resin has achieved the initial enrichment of ivy saponin C. The mixture after flocculation is concentrated into the 50% ethanol eluent, providing a feed solution with fewer impurities and a more concentrated target analyte for subsequent purification by the medium-pressure ODS column.

[0057] S4. Medium-pressure chromatographic purification procedure: Take 50% macroporous resin eluent, concentrate it to half volume, and load it wet-processed. It contains approximately 1g HSC. Further purification is performed using a medium-pressure ODS (30μm, 130 g) column with gradient elution using methanol-water solution as the mobile phase. After equilibrating the column with 10% methanol, wet-processed elution is performed. First, elute with 10% methanol-water solution at 40 mL / min for 1 column volume, then gradually increase the methanol concentration to 60% for 6 column volumes. Analyze the combined target analytes using liquid chromatography to obtain the medium-pressure purified product. The purity calculated by the area normalization method is 80%, and the recovery rate is 85.5%. Real-time monitoring graph of the medium-pressure ODS column purification process is shown below. Figure 6 As shown, the horizontal axis represents the preparation time, and the vertical axis represents the UV detector response signal, reflecting the concentration of components in the eluent. The initial stage corresponds to the 10% methanol column equilibration stage, where the UV signal is at a low level, the column is stable, and no excess components are eluted. The subsequent stage corresponds to the wet loading + 10% methanol wash for one column volume, where the signal rises. After loading, a small amount of unbound impurities are washed by 10% methanol, causing slight fluctuations in the UV signal. Then, the gradient switches to the 60% methanol node, where the UV signal fluctuates, reflecting the gradient elution of different components (including the target analyte HSC). Finally, the stage corresponds to the completion of 6 column volumes of gradient elution, where the signal drops, the target analyte has been eluted and collected, the residual components in the column are reduced, and the signal returns to a stable state. This indicates that the medium-pressure ODS column operates smoothly under this process (flow rate 40 mL / min, gradient elution) without any abnormalities such as column blockage.

[0058] The liquid chromatogram of the combined fractions after medium-pressure preparation is shown below. Figure 7 As shown, the horizontal axis represents the retention time of liquid phase detection, and the vertical axis represents the detector response value. The baseline is flat for most of the retention time intervals in the figure, with only a few small impurity peaks with low response. Only in the later stage does a single main peak with a strong response appear. This main peak corresponds to ivy saponin C. The small impurity peaks are trace impurities that have not been completely removed. This proves that the medium-pressure ODS column effectively removes most of the impurities in the macroporous resin enrichment solution. The main peak is single and has a strong response, indicating that HSC is effectively enriched. This step not only achieves purification but also ensures the recovery rate of the target analyte, providing a high-purity intermediate product for subsequent high-pressure preparation.

[0059] S5. High-pressure preparation and purification step: Load the medium-pressure purified product onto a preparative high-performance liquid chromatography column (10×250mm, 5 μm). High pressure refers to the system working pressure, generally 10-40MPa. Flow rate is 3 mL / min. Detection wavelength is 210 nm. Isocratic elution is performed using acetonitrile-water solution as the mobile phase. Elution is then performed with acetonitrile-water solution with a volume concentration of 28%. The fraction corresponding to the target chromatographic peak is collected to obtain high-purity ivy saponin C.

[0060] Real-time elution monitoring graph of high-pressure preparative liquid chromatography is shown below. Figure 8 As shown, the horizontal axis represents preparation time, and the vertical axis represents detector response value. Real-time elution curves at both 210 nm and 220 nm wavelengths are displayed. Simultaneous dual-wavelength detection of saponins allows for rapid differentiation of target saponin peaks from non-saponin interference peaks without the need for additional standards, enhancing analytical reliability. Pure saponins elute simultaneously at both 210 nm and 220 nm wavelengths, with consistent retention times and peak heights. In contrast, impurities such as sugars and solvents mostly elute at a single wavelength or exhibit significantly different peak response ratios. The main peak shows a sharp, symmetrical peak at the same retention time at both wavelengths, confirming that the peak is ivy saponin C. The absorption of impurities such as sugars and organic acids is concentrated below 210 nm; 220 nm effectively filters these interfering components, improving the accuracy of qualitative judgment. Furthermore, the baseline noise of the 220 nm curve is significantly lower than that of the 210 nm curve, demonstrating effective suppression of interference signals. In practice, the chromatographic peaks corresponding to normal saponins generally exhibit a higher peak height at 210 nm than at 220 nm. The response value of the main peak at 210 nm is significantly higher than that at 220 nm, which is consistent with the typical UV absorption pattern of saponins. If the ratio of peak heights at both wavelengths is abnormal, it suggests the possible presence of co-eluting impurities. In the early stage, corresponding to the baseline before elution of the target analyte, the baseline is extremely flat with only trace amounts of small, low-response impurity peaks. In the later stage, corresponding to the isocratic elution and enrichment of the target analyte, a single, sharp main peak with a strong response appears. This peak is the elution peak of ivy saponin C, and its concentrated shape without dispersed impurity peaks indicates that the elution conditions of high-pressure preparation allow the target analyte to be eluted efficiently and in a concentrated manner. The high-pressure preparation step, based on the product prepared by medium-pressure preparation, further separates the residual trace impurities and separately enriches and elutes the target analyte HSC, providing process assurance for obtaining high-purity products.

[0061] The HPLC purity determination chromatogram of the final product after high-pressure preparation is shown below. Figure 9As shown, the horizontal axis represents retention time, and the vertical axis represents the detection response value. The figure shows only one symmetrical, sharp, strong main peak, with a smooth baseline throughout without any interfering peaks. This peak corresponds to ivy saponin C. The purity of the final product has reached >98%, which is a single, high-purity target compound. This indicates that by flocculation to remove impurities, coarse separation with macroporous resin, medium-pressure chromatography purification, and high-pressure preparation purification, the process of removing impurities, enriching the target compound, and obtaining high-purity HSC from crude Pulsatilla leaf to the final pure product has been successfully achieved.

[0062] This application presents a proprietary extraction and purification process using Pulsatilla chinensis leaves as raw material. This process can reliably yield ivy saponin C with a purity >98%. The process requires no ultra-high pressure or specialized equipment; conventional industrial chromatography equipment is sufficient. The steps are simple, parameters are easily controlled, and complex operations and high costs are avoided. Balancing efficiency and practicality, this process is fully capable of being put into actual production, providing support for the large-scale acquisition of Pulsatilla chinensis saponins.

[0063] <Example 2> The process for extracting ivy saponin C from the leaves of Pulsatilla chinensis includes the following steps: S1. Extraction Procedure: Weigh 1.5 kg of Pulsatilla chinensis leaves, add 14 times the volume of 75% ethanol aqueous solution and reflux for extraction three times: 1.5 h for the first extraction, 1 h for the second, and 1 h for the third. Combine the extracts, concentrate and dry to obtain the extract, dissolve in 20% ethanol aqueous solution, allow to stand and separate into layers, collect the supernatant, concentrate and dry again to obtain a solid powder. The crude extract of Pulsatilla chinensis leaves yielded 316.8 g. Calculated using the external standard two-point method, the HSC content in the original medicinal material was 2.1%, and the HSC content in the crude extract was 9.0%, with an extraction rate of 90.5%. S2. Step-by-step pH-controlled cyclic flocculation: Take 100 g of crude extract of Pulsatilla chinensis leaves and dissolve it in 1 L of 20% ethanol aqueous solution. First, adjust the pH to 4-5 with acid. Add 500 mL of 10% polyaluminum chloride aqueous solution and stir. Adjust the pH in stages with saturated sodium carbonate solution. First, adjust it to 5.5-6.5 to precipitate pectin impurities, and then slowly adjust it to 7-8 to precipitate tannins and pigments. Separate the solid and liquid to obtain the first-stage flocculated liquid and the first-stage flocculated precipitate. Wash the first-stage flocculated precipitate twice with 20% ethanol aqueous solution and resuspend it in 1 L of 20% ethanol aqueous solution. Adjust the pH to 4-5 with citric acid and stir to dissolve the aluminum hydroxide precipitate. Separate the solid and liquid to obtain the acid-treated liquid and the acid-treated precipitate. Adjust the pH of the acid-treated liquid to 7-8 with saturated sodium carbonate solution, stir, and let it stand to separate the solid and liquid to obtain the second-stage flocculated liquid and the second-stage flocculated precipitate. Combine the first-stage flocculated liquid and the second-stage flocculated liquid to obtain the flocculated liquid. Calculations using the two-point external standard method showed that the flocculated liquid contained 8.66 g of HSC, representing a recovery rate of 96.2% relative to the total HSC content in the crude extract (9.0 g). HPLC analysis of the flocculated liquid showed an HSC purity of approximately 22.0%. S3. Macroporous resin enrichment step: Same as in Example 1. Calculated using the two-point external standard method, the macroporous resin enrichment solution contained 7.49 g of HSC, with a recovery rate of 86.5% relative to the loaded sample amount (8.66 g). S4. Medium-pressure chromatography purification steps: Same as in Example 1, the purity calculated by area normalization method is 80.0%, and the recovery rate is 85.5%; S5. High-pressure preparation and purification steps: Same as in Example 1. The purity calculated by the area normalization method is 98.5%, and the recovery rate is 90.0%.

[0064] The HPLC purity determination chromatogram of the final product after high-pressure preparation is shown below. Figure 10 As shown, the horizontal axis represents retention time, and the vertical axis represents the detection response value. There is only one symmetrical, sharp, strong main peak in the figure. The baseline is flat throughout and there are no other peaks. This peak corresponds to ivy saponin C. The purity of the final product has reached >98%, which is a single, high-purity target compound.

[0065] In Example 2, the HSC recovery rate was increased from 83.3% in the original S2 scheme to 96.2% by using the S2 graded pH-controlled cyclic flocculation method. Combined with subsequent chromatographic steps, 5.76 g of high-purity HSC was finally obtained from 100 g of crude extract (containing 9.0 g of HSC), with a total yield of 64.0%, which is significantly improved compared to Example 1 (approximately 55.6%). At the same time, the purity of the final product remained stable at over 98%, achieving a simultaneous improvement in high recovery rate and high purity.

[0066] <Comparative Example 1> The process for extracting ivy saponin C from Pulsatilla chinensis leaves directly follows the conventional process reported in the literature for Pulsatilla chinensis root. The leaves are treated with 70% ethanol for reflux extraction, concentrated until no alcohol odor remains, adsorbed onto AB-8 macroporous resin, eluted with 20% ethanol to remove impurities, and eluted with 50% ethanol to obtain crude extract. The crude extract is then purified by medium- and high-pressure preparative chromatography to obtain ivy saponin C.

[0067] Systematic comparative experiments confirmed that: (1) after concentration or reconstitution of the sample, the viscosity increased significantly, and impurities such as pigments formed a "mud-like" insoluble substance that could not be removed by simple filtration or centrifugation; (2) after loading the sample, the surface of the resin column bed was rapidly contaminated and moved downwards, the elution flow rate was slow, and the eluent was turbid. The macroporous resin was severely contaminated and regeneration was difficult; the purity of the total saponins in the final enriched product was less than 20%, the content of the target saponins was less than 3%, and the total yield was less than 2%.

[0068] The leaves are rich in high molecular weight interfering substances such as chlorophyll, polysaccharides, and flavonoids, which leads to serious technical obstacles in the purification process and causes failure due to changes in the raw material parts. This failure stems from the systematic differences in secondary metabolites in different parts of the plant.

[0069] <Comparative Example 2> The process for extracting ivy saponin C from Pulsatilla chinensis leaves is the same as in Example 1, except that the S2 flocculation and impurity removal step is omitted, meaning the extraction step directly proceeds to the macroporous resin enrichment step.

[0070] <Comparative Example 3> The process for extracting ivy saponin C from Pulsatilla chinensis leaves is the same as in Example 1, except that the S3 macroporous resin enrichment step is omitted, i.e. the flocculation and impurity removal step directly proceeds to the medium-pressure chromatography purification step.

[0071] In Comparative Example 2, the extract was directly loaded onto macroporous resin without flocculation treatment. The resin was competitively adsorbed and contaminated by impurities such as pigments and tannins. The removal rate of pigments and tannins was low, the target stream flowed out, the elution flow rate was slow, the HSC purity of the target component was limited, and the eluent was dark in color and difficult to further purify.

[0072] Comparative Example 3, which only underwent flocculation, sedimentation and washing, could remove some high molecular weight and pigment impurities, but it was insufficient in removing small molecule co-eluting impurities, resulting in low HSC purity. Furthermore, the flocculation process was accompanied by the loss of certain target components.

[0073] Example 1, under specific parameters, sequentially performed flocculation and macroporous resin enrichment. The removal rates of key impurities such as total pigments and tannins were significantly higher than the sum of the removal rates of the two individual steps. Simultaneously, the total recovery rate of the target component HSC was significantly higher than either individual step, purity was greatly improved, the eluent was clear, and the impurity peaks in the HPLC chromatogram were significantly reduced, while the target peak separation was significantly improved. This indicates that S2 flocculation pre-removes interfering substances such as polymers, pigments, and tannins, reducing resin contamination and column bed clogging, significantly improving the sample loading system and adsorption selectivity. S3 macroporous resin efficiently enriches the target component in a clean system, achieving deep purification. The synergistic effect and complementary advantages of these two processes under the specific parameters of this invention produce unexpected technical effects that cannot be achieved by a single step, and cannot be anticipated by those skilled in the art through conventional process combinations.

[0074] <Comparative Example 4> The process for extracting ivy saponin C from Pulsatilla chinensis leaves is the same as in Example 1, except that the S3 macroporous resin enrichment step is performed first, followed by the S2 flocculation and impurity removal step. That is, the extraction step first enters the macroporous resin enrichment step, then the flocculation and impurity removal step, and then the medium-pressure chromatography purification step.

[0075] <Comparative Example 5> The process for extracting ivy saponin C from Pulsatilla chinensis leaves is the same as in Example 1, except that the S2 flocculation and impurity removal step and the S3 macroporous resin enrichment step are omitted. In other words, the extraction step directly proceeds to the medium-pressure chromatography purification step.

[0076] Comparative Example 4, due to the failure to remove strong interfering impurities such as chlorophyll, polysaccharides, and tannins beforehand, resulted in a significant decrease in resin adsorption selectivity, easy column bed contamination and blockage, a dramatic increase in the loss of target components, and a substantial reduction in both purification efficiency and product purity, failing to achieve the expected separation effect.

[0077] Comparative Example 5 directly purified the crude extract using medium-pressure chromatography without flocculation and macroporous resin enrichment. The raw material impurity load was too high and the system was too complex, resulting in extremely low sample loading, severe overlap of chromatographic peaks that could not be separated, rapid column contamination, abnormal increase in column pressure, separation failure, and contamination of chromatographic packing.

[0078] This invention employs a progressive purification route consisting of flocculation to remove impurities, followed by enrichment with macroporous resin, and then purification by medium-pressure chromatography. This is the only feasible technical solution for the high impurity characteristics of Pulsatilla chinensis leaf raw materials. S2 and S3, as essential crude purification steps, play a crucial role in reducing impurities, protecting the column, and improving resolution. They are not simply superpositions or arbitrary combinations of conventional steps in the field; their specific order and combination bring unexpected technical effects.

[0079] <Comparative Example 6> The process for extracting ivy saponin C from Pulsatilla chinensis leaves is the same as in Example 1, except that the S4 purification step is omitted, meaning that the macroporous resin step is followed directly by high-pressure chromatography purification.

[0080] <Comparative Example 7> The process for extracting ivy saponin C from Pulsatilla chinensis leaves is the same as in Example 1, except that the S5 high-pressure purification step, i.e., the product is collected after the medium-pressure chromatographic purification step, is omitted.

[0081] Comparative Example 6, without intermediate-pressure purification, directly underwent high-pressure purification. While the effluent after S3 macroporous resin enrichment achieved preliminary enrichment of saponins, it still contained significant amounts of impurities such as pigments, tannins, polysaccharides, and structurally similar associated saponins. Eliminating S4 and directly proceeding to S5 high-pressure chromatography purification resulted in severe contamination of the expensive preparative chromatography column packing material, leading to a sharp increase in column pressure, rapid decline in column efficiency, and a significant decrease in separation selectivity. This shortened column lifespan, increased equipment maintenance costs, and potentially caused batch-to-batch reproducibility issues due to unstable column efficiency, making it difficult to meet the continuous requirements of industrial production. The S4 impurity removal step is not a dispensable intermediate step, but a necessary bridge connecting crude enrichment and purification. It is crucial for ensuring the technical feasibility, economic rationality, and quality stability of the ivy saponin C preparation process. Eliminating this step would fundamentally undermine the reliability and practicality of the process.

[0082] Comparative Example 7, which did not undergo the final high-pressure purification step, achieved an HSC content of 80%, but the finished product was yellowish in color and contained a small amount of impurities with similar polarity. The high-pressure purification step is a crucial link between intermediate purification and pharmaceutical-grade products, and is a core step in ensuring the safety, efficacy, and quality control of ivy saponin C; it cannot be omitted.

[0083] Medium-pressure preparative chromatography typically uses packing materials with particle sizes greater than 10 μm and operates within a low to medium pressure range of 0.5–5 MPa. Due to the larger particle size and relatively lower column bed density, the mass transfer resistance of the mobile phase within the column is low, allowing for higher flow rates and larger injection volumes (generally 1%–5% of the column volume), making it suitable for the coarse separation and enrichment of gram- to hundred-gram samples. However, its theoretical plate number is only 1000–3000, limiting its separation capacity and making it difficult to achieve fine separation of compounds with similar structures. High-pressure preparative chromatography, on the other hand, uses packing materials with particle sizes of 5–10 μm and smaller, operating at 10–30 MPa. The smaller particle size significantly increases the specific surface area, providing more interaction sites, enabling theoretical plate numbers to reach over 10,000, resulting in a substantial improvement in separation resolution. It can simultaneously achieve separation purity and processing efficiency, making it suitable for the high-purity purification of milligram- to gram-level samples and a key technology for obtaining monomeric compounds and meeting stringent quality standards. In general, medium-pressure preparative chromatography sacrifices processing efficiency for scale-up, while high-pressure preparative chromatography sacrifices separation purity for increased pressure. They share a common separation mechanism and complement each other in terms of engineering implementation and product purity levels. Using them in combination ensures a stable and reliable preparation process, thereby achieving stable and controllable acquisition of the target product HSC.

[0084] The number of devices and processing scale described herein are for the purpose of simplifying the description of the invention. Applications, modifications, and variations of the invention will be readily apparent to those skilled in the art.

[0085] Although embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the specification and embodiments. They can be applied to various fields suitable for the present invention. For those skilled in the art, other modifications can be easily made. Therefore, without departing from the general concept defined by the claims and their equivalents, the present invention is not limited to the specific details and illustrations shown and described herein.

Claims

1. A process for extracting ivy saponin C from the leaves of Pulsatilla chinensis, characterized in that, Includes the following steps: S1. Extraction steps: The leaves of Pulsatilla chinensis were extracted by reflux with an ethanol-water solution. The extract was then concentrated and dried to obtain crude extract of Pulsatilla chinensis leaves. S2, Flocculation and Impurity Removal Step: Dissolve the crude extract of Pulsatilla chinensis leaves in an ethanol aqueous solution, add flocculant for flocculation treatment and adjust the pH to 7-8, and obtain the flocculated liquid after solid-liquid separation; S3. Macroporous resin enrichment step: The flocculated liquid is loaded onto a macroporous resin column and eluted with ethanol-water solution as the mobile phase. The eluted portion rich in ivy saponin C is collected to obtain the macroporous resin enrichment solution. S4. Medium-pressure chromatographic purification step: Load the macroporous resin enrichment solution onto a reversed-phase silica gel preparative chromatographic column, elute with methanol-water solution as the mobile phase, collect the target fraction, and obtain the medium-pressure purified product. S5. High-pressure preparation and purification step: The medium-pressure purified product is loaded onto a preparative high-performance liquid chromatography column, eluted with acetonitrile-water solution, and the fraction corresponding to the target chromatographic peak is collected to obtain high-purity ivy saponin C.

2. The process for extracting hederaponin C from the leaves of Pulsatilla chinensis according to claim 1, characterized in that, In step S1, the leaves of Pulsatilla chinensis are refluxed and extracted 1-3 times using an ethanol aqueous solution with a volume concentration of 70% to 80%. The extracts are combined, concentrated and dried to obtain the extract, which is then dissolved in an ethanol aqueous solution with a volume concentration of 15% to 25%. After standing and separating into layers, the supernatant is collected, concentrated and dried again to obtain the crude extract of Pulsatilla chinensis leaves.

3. The process for extracting hederaponin C from the leaves of Pulsatilla chinensis according to claim 1, characterized in that, In step S2, the flocculant is polyaluminum chloride. The crude extract of Pulsatilla chinensis leaves is dissolved in an ethanol aqueous solution with a volume concentration of 15% to 25%. Polyaluminum chloride is added for flocculation treatment and the pH is adjusted to 7-8. After solid-liquid separation, the precipitate obtained by separation is washed with an ethanol aqueous solution with a volume concentration of 15% to 25%. The washing liquid is combined with the liquid obtained by solid-liquid separation to obtain the flocculated liquid.

4. The process for extracting hederaponin C from the leaves of Pulsatilla chinensis according to claim 3, characterized in that, In step S2, the flocculant is polyaluminum chloride, and the flocculation treatment employs staged pH-controlled circulating flocculation, specifically including: S21. The crude extract of Pulsatilla chinensis leaves dissolved in an ethanol aqueous solution with a volume concentration of 15% to 25% was first adjusted to pH 4 to 5 with acid, polyaluminum chloride was added, and after stirring and reacting, the pH was adjusted to 7-8 in stages with alkaline solution. After standing, aluminum hydroxide precipitate was fully generated, and solid-liquid separation was performed to obtain the first-stage flocculated liquid and the first-stage flocculated precipitate. S22. After washing the first-stage flocculation precipitate with an ethanol aqueous solution with a volume concentration of 15% to 25%, it is resuspended in an ethanol aqueous solution with a volume concentration of 15% to 25%. The pH is adjusted to 4 to 5 with citric acid, and the aluminum hydroxide precipitate is dissolved by stirring. After standing, the solid and liquid are separated to obtain the acid treatment solution. S23. Adjust the pH of the acid treatment solution to 7-8 with alkaline solution, let it stand to allow aluminum hydroxide precipitate to form fully, and then separate the solid and liquid after standing to obtain the second-stage flocculated liquid. S24. Combine the first-stage flocculated liquid with the second-stage flocculated liquid to obtain the flocculated liquid.

5. The process for extracting hederaponin C from the leaves of Pulsatilla chinensis according to claim 1, characterized in that, The elution in step S3 is gradient elution, first eluting with an ethanol-water solution with a volume concentration of 15% to 25% for 3 to 5 column volumes, and then eluting with an ethanol-water solution with a volume concentration of 45% to 55% for 4 to 6 column volumes.

6. The process for extracting hederaponin C from the leaves of Pulsatilla chinensis according to claim 1, characterized in that, In step S3, the macroporous resin column is pretreated before use. The pretreatment includes soaking in ethanol and then washing with water until there is no alcohol odor.

7. The process for extracting hederaponin C from the leaves of Pulsatilla chinensis according to claim 1, characterized in that, The macroporous resin in step S3 is one of type D101, type LX-20B, and type AB-8.

8. The process for extracting hederaponin C from the leaves of Pulsatilla chinensis according to claim 1, characterized in that, The elution in step S4 is a gradient elution, first eluting with a methanol-water solution with a volume concentration of 8% to 12% for 0.5 to 1.5 column volumes, and then gradually increasing the methanol concentration to 55% to 65% for 5 to 7 column volumes.

9. The process for extracting hederaponin C from the leaves of Pulsatilla chinensis according to claim 1, characterized in that, The elution in step S5 is isocratic elution, using an acetonitrile-water solution with a volume concentration of 25% to 30%.

10. The process for extracting hederaponin C from the leaves of Pulsatilla chinensis according to claim 1, characterized in that, In step S5, the medium-pressure purified product is concentrated to obtain a solid. The solid is then dissolved in methanol and loaded onto a preparative high-performance liquid chromatography column.

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