Cooked pseudo-ginseng and preparation method thereof

Abstract

The invention provides cooked pseudo-ginseng and a preparation method thereof, and belongs to the technical field of traditional Chinese medicine processing. The preparation method comprises the following steps: crushing raw pseudo-ginseng, and mixing and infiltrating with water to obtain moistened pseudo-ginseng; steaming and drying the moistened pseudo-ginseng to obtain cooked pseudo-ginseng; the particle size of crushing is 2-4 mm, and the infiltration time is 2 h. According to the invention, the tissue structure of panax notoginseng is effectively softened by adopting moisture infiltration, and the cell wall permeability and thermal conversion efficiency of ginsenoside are remarkably improved by utilizing a synergistic effect mechanism between particle size reduction and optimized moisture infiltration. The specific steaming temperature and time balance the conversion rate and the potential thermal degradation risk, the maximum accumulation of the target rare saponin is realized, and the yield of Rk3, Rh4, 20 (S)-Rg3 and 20 (R)-Rg3 is obviously improved. The'moisture activation-gradient temperature control 'strategy is synergistically optimized from physical and chemical kinetics levels, and is a core mechanism for solving the problem of large batch difference and improving the uniformity and activity of cooked pseudo-ginseng products.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of traditional Chinese medicine processing, and particularly relates to a prepared panax notoginseng and a preparation method thereof. BACKGROUND

[0002] Panax notoginseng, a precious medicinal material of the Araliaceae family, has a long history of clinical application in traditional Chinese medicine. Its medicinal value can be traced back to the Ming Dynasty's Compendium of Materia Medica. It is famous for its characteristics of "raw dispersing and cooked invigorating" - raw products disperse blood stasis and stop bleeding, and cooked products invigorate the body and strengthen the body, which is a classic example of the theory of "raw and cooked different treatment" of traditional Chinese medicine. Modern pharmacological studies have confirmed that the bioactive substances of panax notoginseng are mainly saponins, with more than 150 kinds, mainly including protopanax diol (PPD type, such as Rb1, Rd) and protopanax triol (PPT type, such as Rg1, Re) saponins. These components play the roles of hemostasis, anti-inflammatory and cardiovascular protection by regulating key signaling pathways such as PI3K / AKT and NF-κB.

[0003] The process of processing raw panax notoginseng triggers a series of chemical transformations, significantly changing its pharmacodynamic basis and pharmacological effects, which is the material basis of its "raw and cooked different treatment" characteristics. High-temperature processing catalyzes the deglycosylation, dehydration and isomerization of original saponins, generating a series of rare saponins with stronger or unique biological activity. G-Rk3, G-Rh4, 20( S )G-Rg3 and 20( R )G-Rg3 are the main degradation products formed under high temperature / high humidity steaming conditions through a specific pathway, and their concentrations are directly related to the processing intensity and efficiency. These rare saponins each have significant and unique biological activities related to the invigorating effect of cooked panax notoginseng. For example, 20( S )-Rg3 induces tumor cell apoptosis (IC50=12.3 μM) by inhibiting the AKT / mTOR pathway, showing significant anti-tumor effect. Its epimer 20( R )-Rg3 has obvious neuroprotective effect, and can significantly improve the survival rate of neurons (up to 37.2%) in an Aβ25-35-induced neuronal injury model. Rk3 and Rh4 also contribute to the overall anti-inflammatory, antioxidant, blood-enriching and potential anticancer activity of cooked panax notoginseng.

[0004] Although the pharmacodynamic basis of cooked panax notoginseng is increasingly clear, its industrialization is still subject to the volatility of the production process, and the traditional processing parameters (particle size, soaking conditions, steaming temperature and time) lack systematic optimization based on saponin transformation kinetics. Studies have shown that the conversion rate is highly sensitive to these parameters. For example, excessive grinding poses a risk of degradation of heat-sensitive components. Insufficient water soaking cannot fully activate the transformation process, and suboptimal temperature and time combinations can lead to incomplete transformation or degradation, resulting in low total yield of key rare saponins. These inconsistencies directly affect the efficacy and safety of the product. SUMMARY

[0005] Therefore, the present application aims to provide a prepared panax notoginseng and a preparation method thereof to solve the problems of low yield and significant batch-to-batch difference of key rare saponins Rk3, Rh4, 20(Rg3) and 20(Rg3) in prepared panax notoginseng due to fluctuation of processing parameters. S )-Rg3. R )-Rg3.

[0006] In order to achieve the above-mentioned application purposes, the present application provides the following technical solutions. The present application provides a preparation method of prepared panax notoginseng, comprising the following steps: 1) crushing raw panax notoginseng and mixing with water for soaking to obtain soaked panax notoginseng; 2) steaming and drying the soaked panax notoginseng to obtain prepared panax notoginseng; The particle size of the crushing is 2-4 mm, and the soaking time is 2 h.

[0007] Preferably, in step 1), the temperature of the water is 24.5-25.5℃, the mass ratio of the raw panax notoginseng to water is 1:1, and the moisture content of the raw panax notoginseng after soaking is 100% of the total mass of the material.

[0008] Preferably, in step 1), the raw panax notoginseng is dried before crushing, the drying temperature is 50-55℃, the drying time is 48-52 h, and the moisture content after drying is ≤8%.

[0009] Preferably, in step 2), the steaming temperature is 100-130℃, and the steaming time is 3-6 h.

[0010] Preferably, in step 2), the drying temperature is 58-62℃, the drying time is 4-5 h, and the moisture content after drying is ≤9%.

[0011] The present application also provides the prepared panax notoginseng prepared by the preparation method.

[0012] Compared with the prior art, the present application has the following beneficial effects: The present application effectively softens the structure of panax notoginseng by water soaking (100% w / w), significantly improves the cell wall permeability and thermal conversion efficiency of ginsenosides by the synergistic mechanism between particle size reduction (2-4 mm) and optimized water soaking, accurately matches the activation energy (Ea≈58.3 kJ / mol) required for deglycosylation and dehydration reaction of ginsenosides (such as Rb1 and Rd) by precise steaming temperature, ensures efficient reaction, balances the conversion rate and potential thermal degradation risk by specific steaming time window, and maximizes the accumulation of target rare saponins, thereby significantly improving the yield of Rk3, Rh4, 20(Rg3) and 20(Rg3). S) -Rg3 and 20 ( R The yield of Rg3. The “moisture activation-gradient temperature control” strategy is synergistically optimized from the physical and chemical dynamics level, and is the core mechanism for solving the large batch difference and improving the uniformity and activity of processed panax notoginseng products. BRIEF DESCRIPTION OF DRAWINGS

[0013] Figure 1 is a single factor influence experiment of processing technology (wherein, A is the influence of three seven granules infiltration pretreatment on the content of four saponins after processing; B is the influence of three seven granules particle size on the content of four saponins after processing; C is the influence of different steaming time on the content of four saponins after processing; D is the influence of different steaming temperature on the content of four saponins after processing); Figure 2 is four ginsenosides Rk3, Rh4, 20 ( S ) -Rg3 and 20 ( R ) Rg3 standard curve; Figure 3 is the HPLC chromatogram of mixed reference solution and processed panax notoginseng extract (wherein, A is the mixed reference solution, B is the processed panax notoginseng extract, 1 is ginsenoside Rk3, 2 is ginsenoside Rh4, 3 is ginsenoside 20 ( S ) -Rg3, 4 is ginsenoside 20 ( R ) -Rg3). DETAILED DESCRIPTION

[0014] The application provides a preparation method of processed panax notoginseng, which comprises the following steps: 1) crushing raw panax notoginseng, mixing and infiltrating with water to obtain infiltrated panax notoginseng; 2) steaming and drying the infiltrated panax notoginseng to obtain processed panax notoginseng; The particle size of the crushing is 2-4 mm, and the infiltration time is 2 h.

[0015] In the application, the drying before crushing is carried out, the drying temperature is preferably 50-55 DEG C, the drying time is preferably 48-52 h, further preferably 50 h, the drying is carried out to the water content of less than or equal to 8%, the water temperature is preferably 24.5-25.5 DEG C, further preferably 25 DEG C, the mass ratio of raw panax notoginseng to water is 1:1, the water content in the raw panax notoginseng after infiltration accounts for 100% of the total mass of the substance, the steaming temperature is preferably 100-130 DEG C, further preferably 120 DEG C, the steaming time is preferably 3-6 h, further preferably 5 h, the drying temperature is preferably 58-62 DEG C, further preferably 60 DEG C, the drying time is preferably 4-5 h, further preferably 4.5 h, and the drying is carried out to the water content of less than or equal to 9%.

[0016] The technical solutions provided by the present application are described in detail below in conjunction with examples, but they should not be understood as limiting the scope of protection of the present application.

[0017] Materials used in the present application are as follows: Chromatographic grade solvents (methanol and acetonitrile, HPLC grade) were purchased from Fisher Scientific (Fair Lawn, NJ, USA). Ultrapure water was prepared using a Millipore system (Millipore, Bedford, MA, USA), and the source of pure water was Wahaha Group (Hangzhou, China). Ginsenoside standards: G-Rk3 and G-Rh4 were from Stanford Analytical Chemistry Corporation (Stanford, CA, USA); 20 (G-Rg3) was provided by Chengdu GLP Biotechnology Co., Ltd. (Chengdu, China); 20 (G-Rg3) was purchased from the National Institute for Food and Drug Control (NIFDC, Beijing, China). Purity verification: all standards were verified by HPLC peak area normalization, and the purity was greater than 98%. Standard information is shown in Table 1. S ) G-Rg3 was provided by Chengdu GLP Biotechnology Co., Ltd. (Chengdu, China); 20 (G-Rg3) was purchased from the National Institute for Food and Drug Control (NIFDC, Beijing, China). Purity verification: all standards were verified by HPLC peak area normalization, and the purity was greater than 98%. Standard information is shown in Table 1. R

[0018] Table 1 Standard information

[0019] The four standards were verified by HPLC peak area normalization method, and the purity was greater than 98%. All other chemical reagents were purchased from Beijing Chemical Plant (Beijing, China) and were all analytical pure. All notoginseng was purchased from Wenshan Zhuang and Miao Autonomous Prefecture, Yunnan Province, China. Sample identification was based on the macroscopic and microscopic examination of Chinese Pharmacopoeia (2025 Edition).

[0020] Example 1

[0021] Sixty notoginseng roots were selected according to the agricultural grading standard, ensuring uniform maturity and complete structure. Fresh roots were air-dried at 53°C for 50 h to make the moisture content ≤8%, and then mechanically pulverized into coarse powder with a hammer mill. The powder was sieved through a No. 4 sieve (nominal aperture: 4.0 mm), and particles with a diameter of 2-4 mm were separated, and the fragments with too small or too large size were discarded. The sieved particles were soaked in pure water (100% w / w) at 25°C for 2 h to achieve uniform distribution of moisture. Then, the material was steamed in a layered tray (≤3 cm high, covered with bamboo mat) at a controlled temperature of 120°C for 5 h. After steaming, the material was dried at a temperature of 60°C for 4.5 h until the moisture content was reduced to below 9%.

[0022] Example 2

[0023] ​Sixty roots of Panax notoginseng were selected according to the agricultural grading standard to ensure uniform maturity and structural integrity. Fresh roots were air-dried at 50 °C for 48 h to make the moisture content ≤8%, and then mechanically pulverized into coarse powder with a hammer mill. The powder was sieved through a No. 4 sieve (nominal aperture: 4.0 mm) to separate out the particles with a diameter of 2-4 mm, and the fragments with size too small or too large were discarded. The sieved particles were soaked in purified water (100% w / w) at 24.5 °C for 2 h to achieve uniform distribution of moisture. Then the material was steamed at a controlled temperature of 100 °C for 6 h in layered trays (≤3 cm high, covered with bamboo mats). After steaming, the material was dried at a temperature of 58 °C for 4 h until the moisture content was reduced to below 9%.

[0024] Example 3

[0025] Sixty roots of Panax notoginseng were selected according to the agricultural grading standard to ensure uniform maturity and structural integrity. Fresh roots were air-dried at 55 °C for 52 h to make the moisture content ≤8%, and then mechanically pulverized into coarse powder with a hammer mill. The powder was sieved through a No. 4 sieve (nominal aperture: 4.0 mm) to separate out the particles with a diameter of 2-4 mm, and the fragments with size too small or too large were discarded. The sieved particles were soaked in purified water (100% w / w) at 25.5 °C for 2 h to achieve uniform distribution of moisture. Then the material was steamed at a controlled temperature of 130 °C for 3 h in layered trays (≤3 cm high, covered with bamboo mats). After steaming, the material was dried at a temperature of 62 °C for 5 h until the moisture content was reduced to below 9%.

[0026] Experimental Example 1

[0027] Optimization of processing technology

[0028] 1. Pre-soaking treatment of Panax notoginseng

[0029] To investigate the effects of pre-soaking treatment on cooking efficiency and ginsenoside conversion, four different particle sizes of Panax notoginseng roots were prepared using a combination of mechanical grinding and standardized sieving: The dried Panax notoginseng roots were mechanically ground into coarse powder using a hammer mill; the powder was then sequentially sieved through a series of standard sieves (No. 4, No. 10, No. 20, and No. 40) to separate four particle size groups: 8-10 mm (retained on No. 4 sieve); 5-8 mm (passed through No. 4 sieve, retained on No. 10 sieve); 2-4 mm (passed through No. 10 sieve, retained on No. 20 sieve); and 1-2 mm (passed through No. 20 sieve, retained on No. 40 sieve). Sixty whole roots and particles from each of the four particle size fractions (n=3 per group, 50 g per group) were weighed and transferred to labeled 500 mL Erlenmeyer flasks. Each flask was filled with 400 mL of ultrapure water, shaken vigorously for 1 minute, and equilibrated at 25°C. At predetermined time intervals (1, 2, 4, 6, 8, 24, 34, 48, 58, 62 hours), the supernatant was filtered, and volume changes were recorded. Two to three particles were randomly selected from each flask, halved, and the hydration of the central tissue was examined. Ginsenoside Rk3, ginsenoside Rh4, and ginsenoside 20- ( S Rg3 and ginsenoside 20- ( R The Rg3 content was used as the evaluation index (the method for determining the ginsenoside content is the same as in Experiment 2) to confirm the most suitable treatment.

[0030] Experimental results: The soaking time for intact main roots of 60 Panax notoginseng plants was 58-62 hours, with water absorption of about 95% of their own weight. The soaking time for Panax notoginseng granules with a particle size of 8-9 mm was 24 hours, with water absorption of about 97.5% of their own weight. The soaking time for Panax notoginseng granules with a particle size of 4-5 mm was 2 hours, with water absorption of 100% of their own weight.

[0031] (2) Take two portions of Panax notoginseng granules with a particle size of 2-4 mm. Soak one portion in purified water at its own weight for 2 hours, and leave the other portion untreated. Steam at 120℃ for 5 hours, using ginsenoside Rk3, ginsenoside Rh4, and ginsenoside 20 ( S Rg3 and ginsenoside 20 ( R The Rg3 content was used as the evaluation index (the method for determining the ginsenoside content is the same as in Experiment 2) to compare the effects of soaking on the content of four saponins in processed Panax notoginseng granules.

[0032] Experimental results: such as Figure 1 As shown in Table 2.

[0033] Table 2 Results of Single-Factor Experiments on Processing Technology

[0034] Table 2 shows that soaking the Panax notoginseng granules before steaming significantly increased the content of the four saponins. After soaking the 2-4 mm granules in 100% purified water for 2 hours, the content of 20 (…)…R The content of α-Rg3 was 220% higher than that of the unsoaked group (p<0.01), confirming that sufficient soaking can promote saponin conversion. The mechanism may be related to water activation and changes in cell wall structure. Therefore, pretreatment by soaking before steaming Panax notoginseng is essential.

[0035] 2. Particle size screening

[0036] To investigate the effects of particle size on cooking efficiency and ginsenoside conversion, four different particle size groups were prepared using a combination of mechanical grinding and standardized sieving: 8-10 mm; 5-8 mm; 2-4 mm; and 1-2 mm. Each batch was pretreated by soaking in purified water equivalent to 100% of its mass: 1-2 mm and 2-4 mm particles were soaked at 25±0.5°C for 2 hours, while 5-8 mm and 8-10 mm particles required 24 hours to reach saturation. After cooking, the materials were dried at 60±2°C for 4-5 hours, continuing drying until the moisture content dropped below 9%. Ginsenosides Rk3, Rh4, and 20-( S Rg3 and ginsenoside 20- ( R The Rg3 content was used as the evaluation index (the method for determining the ginsenoside content is the same as in Experiment 2) to confirm the most suitable particle size.

[0037] Experimental results: such as Figure 1 As shown in Table 2, reducing particle size significantly prolonged grinding time and increased material loss, with the lowest processing yield observed in the 1-2 mm particle size. This loss was mainly due to adhesion during processing. Simultaneously, particle size was negatively correlated with ginsenoside content, with smaller particles (e.g., 1-2 mm) producing higher concentrations of all four ginsenosides compared to larger particles (8-10 mm) (p < 0.05). Notably, although the ginsenoside content was slightly higher in 1-2 mm particles, 2-4 mm particles showed the optimal balance between bioactive yield and production efficiency, minimizing processing losses while achieving high ginsenoside yields: G-Rk3 (8.5118 mg / g), G-Rh4 (11.4030 mg / g), and 20 (…). S G-Rg3 (7.4440 mg / g) and 20 ( R G-Rg3 (4.5756 mg / g) was used to determine the particle size range as 2-4 mm.

[0038] 3. Optimization of steaming parameters

[0039] Panax notoginseng granules (2-4 mm) were initially hydrated in ultrapure water (granule to water mass ratio 1:1) at 25±0.5°C for 2 h. Subsequently, the hydrated granules were exposed to autoclave for 3 h under controlled steam conditions at gradient temperatures (100, 110, 120, and 130°C). After steaming, the material was dried at 60±2°C for 4-5 h until the moisture content dropped below 9%. All heat treatments were repeated three times (n=3) to ensure reproducibility. Ginsenoside Rk3, ginsenoside Rh4, and ginsenoside 20- ( S Rg3 and ginsenoside 20- ( R The Rg3 content was used as the evaluation index (the method for determining the ginsenoside content is the same as in Experiment 2) to confirm the most suitable steaming temperature.

[0040] Experimental results: such as Figure 1 As shown in Table 2, increasing temperature significantly reduced the yield (100°C: 96.10% → 130°C: 90.41%, p < 0.01), which is due to the accelerated deglycosylation of protopanaxadiol-type saponins (such as Rb1 and Rd) via β-elimination. Most importantly, at 100°C, 20 (… S G-Rg3 or 20 ( R G-Rg3 and G-Rk3 / G-Rh4 were also scarce. At 110°C, the total ginsenoside content reached 16.5101 mg / g [G-Rk3: 4.7026 mg / g, G-Rh4: 6.4943 mg / g, 20(S)G-Rg3: 3.3625 mg / g, 20(R)G-Rg3: 1.9507 mg / g]. Cooking at 120°C maximized the conversion rate of ginsenosides, increasing the total content by 78.6% (p<0.01) to 35.4777 mg / g. The content of each ginsenoside was 1.81, 1.76, 2.21, and 2.35 times higher than at 110°C, respectively. Although 130°C further increased the content by 18.3% (p>0.05), diminishing returns and safety constraints (equipment limit: 134°C) determined that 120°C was the optimal temperature for balancing saponin yield, energy efficiency and operational safety.

[0041] 4. Steaming time assessment

[0042] Panax notoginseng granules (2-4 mm) were initially hydrated in ultrapure water (granule to water mass ratio 1:1) at 25±0.5°C for 2 hours. Subsequently, the hydrated granules underwent controlled steam treatment at 120°C for gradient durations (3, 4, 5, and 6 hours) using an autoclave. After steaming, the material was dried at 60±2°C for 4-5 hours until the moisture content dropped below 9%. All heat treatments were repeated three times (n=3) to ensure reproducibility. Ginsenoside Rk3, ginsenoside Rh4, and ginsenoside 20- ( S Rg3 and ginsenoside 20- ( R The Rg3 content was used as the evaluation index (the method for determining the ginsenoside content is the same as in Experiment 2) to confirm the most suitable steaming time.

[0043] Experimental results: such as Figure 1 As shown in Table 2, extending the cooking time reduced the yield (p>0.05) but increased the ginsenoside content. Steaming for 3 hours: G-Rk3, G-Rh4, 20 ( S G-Rg3, 20 ( R The contents of ginsenosides G-Rg3 were 8.5118, 11.4030, 7.4440, and 4.5756 mg / g, respectively; after steaming for 4 h, the contents increased by 6.84%, 8.00%, 10.98%, and 14.25% compared to 3 h (p<0.01); after steaming for 5 h, 20(S)G-Rg3 and 20(R)G-Rg3 increased significantly (4.23% and 4.08%, p<0.05), while G-Rk3 and G-Rh4 remained stable (±0.50%); after steaming for 6 h, all ginsenosides increased (G-Rk3: +2.62%; G-Rh4: +3.32%; 20( S G-Rg3: +6.94%; 20 ( R G-Rg3: +7.17% compared to 5h, p<0.05), but diminishing returns (lower efficacy per unit time). Crucially, extending the steam period beyond 5h provided marginal benefits (e.g., 20...). S (G-Rg3 increased by less than 5% from 5h to 6h), but this resulted in disproportionate energy costs. Therefore, 5h was determined to be the optimal time to balance ginsenoside yield and process efficiency.

[0044] Experimental Example 2

[0045] 1. Preparation of standard solutions

[0046] G-Rk3, Rh4 and 20 ( SAccurately weigh 40 mg of each G-Rg3 standard and dissolve them in methanol in 25 mL flasks to prepare concentrations of 1.5570 mg / mL (G-Rk3), 1.5605 mg / mL (G-Rh4), and 1.4719 mg / mL (20 mg / mL). S The stock solution of G-Rg3. Additionally, 20 ( R G-Rg3 (20 mg) was dissolved in methanol in a 100 mL volumetric flask to obtain a stock solution of 0.17114 mg / mL. Equal portions of G-Rk3, G-Rh4, and 20 mg / mL were then dissolved in methanol. S Transfer 1 mL of each of the G-Rg3 stock solution to a 10 mL volumetric flask, and mix with 5 mL of 20 ( R The G-Rg3 stock solution was mixed and diluted with 60% ethanol to the desired volume, yielding final concentrations of 0.1557 mg / mL (G-Rk3), 0.1561 mg / mL (G-Rh4), and 0.1472 mg / mL (20 mg / mL). S A mixed working solution of G-Rg3. 0.0856 mg / mL (20 ( R (G-Rg3). Before injection, all diluted solutions were filtered through a 0.22 μm polyvinylidene fluoride (PVDF) membrane (Millipore, Billerica, MA, USA) to remove particulate contaminants. The filtrate was used for HPLC analysis, and a standard curve was plotted (e.g., G-Rg3). Figure 2 (As shown in the table). Specific concentrations for preparing the standard solutions are detailed in Table 3.

[0047] Table 3. Concentration Table for Standard Solution Preparation (mg / ml)

[0048] Depend on Figure 2 It can be seen that, based on the HPLC results of six standards with different concentrations, the functional relationship between the injection volume (X) and the peak area (Y) was obtained. The results show that Rk3, Rh4, and 20 ( S -Rg3 and 20 ( R )-Rg3 of R 2 All values ​​are greater than 0.9997, indicating a good linear relationship.

[0049] 2. Sample preparation

[0050] The steam processing scheme of Panax notoginseng was systematically optimized using response surface methodology (RSM). The three key factors were: steam temperature (120°C, ±0.5°C), duration (5 h, ±5 min), and humidification rate (100% w / w, ±0.2%).

[0051] Following the method described in Example 1, industrial-scale validation was conducted at Yunnan Baiyao Group Wenshan Qihua Co., Ltd. (Wenshan, China) according to GMP standards, producing three batches (S20230401, S20231113, S20240201) of processed Panax notoginseng, with full traceability throughout the process. 0.6 g of the prepared processed Panax notoginseng powder was accurately weighed (accuracy: ±0.1 mg) and placed in a round-bottom flask. Reflux extraction: 50 mL of 60% (v / v) ethanol was added, and the total weight was recorded. The mixture was refluxed in an 80°C water bath for 1 h. Cooling and replenishment: After cooling to room temperature, fresh 60% ethanol was added to compensate for weight loss due to solvent evaporation. For homogeneity, the mixture was vortexed. Filtration: The extract was filtered through a 0.22 μm PVDF membrane to remove particulate matter, and then injected by HPLC.

[0052] 3. Quantitative analysis by HPLC

[0053] The processed Panax notoginseng sample was analyzed by HPLC using an Agilent 1260 series high-performance liquid chromatograph equipped with a quaternary pump, autosampler, column temperature chamber, and diode array detector (DAD). Chromatographic column: Agilent ZORBAX-SB-C18 (250 mm × 4.6 mm, 5 μm); mobile phase: acetonitrile-water (40:60, v / v) isocratic elution; flow rate: 1.0 mL / min; column temperature: 25℃; detection wavelength: 203 nm; injection volume: 10 μL; theoretical plate number was set according to ginsenoside 20 (… R The calculated Rg3 peak value should be no less than 6000. The HPLC chromatograms of the standard compound and the sample are shown below. Figure 3 As shown.

[0054] 4. Validation of HPLC analytical method

[0055] The rationality of the HPLC analytical method was verified through evaluations of specificity, limit of detection (LOD), limit of quantitation (LOQ), linearity and range, precision, stability, repeatability, and accuracy. The verification procedure was designed as follows: LOD: signal-to-noise ratio method (S / N≥3, N=3); LOQ: determined by three times the LOD concentration, ensuring a signal-to-noise ratio (S / N) >10; linearity and range: accurate extraction of ginsenosides G-Rk3, G-Rh4, and 20 ( S G-Rg3 and 20 ( RAliquots (2, 4, 6, 8, 10, and 12 μL) of the mixed reference standard solution G-Rg3 were injected into the HPLC system under the specified chromatographic conditions of the profile chromatography system (volume was directly adjusted to avoid analyte loss due to dilution). Precision (intra-day): The same test solution was analyzed six times consecutively within one day to evaluate intra-day precision. Precision (inter-day): The same sample was measured six times daily over three consecutive days, and inter-day precision was calculated using comprehensive multi-day data. Repeatability: Six independent sample formulations from the same batch were analyzed in parallel to assess repeatability. Accuracy: Accuracy was determined by standard spiking recovery experiments. A known amount of reference standard (in a 1:1 ratio with the target analyte) was added to the test solution, and the recovery rate (%) was calculated. Solution stability: The stability of the sample solution was evaluated at 0, 2, 4, 8, 12, 16, 20, 24, and 36 hours after preparation under controlled storage conditions.

[0056] Experimental results are shown in Table 4. The LOD and LOQ ranged from 1.50 to 5.28 µg / mL and 4.49 to 15.9 µg / mL, respectively.

[0057] Table 4 Methodological Validation of HPLC Analytical Methods

[0058] RSD: Relative Standard Deviation.

[0059] Table 4 shows that the validation results of the method, expressed as the relative standard deviation (RSD) of the contents of the four ginsenosides, have high reliability; the lower the RSD value, the higher the precision. For repeatability assessment, the RSD values ​​of all target peaks in six replicate analyses of the same sample were all below 0.38%, and the precision (daytime) RSD values ​​of all target peaks were all below 1.8%. The RSD values ​​of the four ginsenoside contents measured at multiple time points within 36 hours at room temperature, all below 1.2%, confirmed the stability of the sample. The repeatability of the method was validated using six independently prepared samples from the same batch, with RSD values ​​of all peaks below 1.0%. Under known standard concentrations, the spiked recoveries of the four major saponins were evaluated, with recoveries ranging from 91.95% to 101.34% and RSDs ranging from 0.93% to 1.8%.

[0060] 5. Establishment of QAMS analysis method

[0061] ① Establishment of a multi-component quantitative analysis and evaluation model using the single-label method (QAMS)

[0062] In this invention, ginsenoside 20 ( R20-Rg3 is the most stable and readily available of the saponins in processed Panax notoginseng. It is easily separated under conventional chromatographic conditions, the separation technology is mature, and its acquisition cost is relatively low. It also exhibits significant activity in various aspects, including anticancer, immune enhancement, antioxidant, anti-inflammatory, and cardiovascular protection. Therefore, 20-Rg3... R Rg3 is the most suitable internal standard for the determination of Panax notoginseng content.

[0063] ② Relative correction factor

[0064] The core step in establishing the QAMS method is to achieve multi-component quantification by calculating the relative correction factor (RCF) between the internal reference and the target component. The specific steps are as follows: Preparation of standard solution: Accurately pipette the mixed standard solution (containing ginsenosides Rk3, Rh4, and 20) S )-Rg3 and 20-( R )-Rg3)2, 4, 6, 8, 10, 12 μL, and analyzed according to the above chromatographic conditions.

[0065] RCF calculation: with 20 ( R Rg3 is an internal reference. The RCF values ​​of each component are calculated according to formula (1): (1) In formula (1), F 20(R)Rg3 / mi The target object (mi=Rk3 / Rh4 / 20) S )-Rg3) relative to internal reference 20 ( R The correction factor for -Rg3; A 20（R）Rg3 C 20（R）Rg3 20 (of the mixed reference standards) R Peak area and concentration of )-Rg3.

[0066] Experimental results are shown in Table 5.

[0067] Table 5. Relative Correction Factors for Saponins

[0068] Table 5 shows that within the validated linear range, saponin content is directly proportional to the chromatographic peak area. Changes in injection volume alter both saponin content and detector response; however, the relative correction factor (RCF) theoretically remains constant. The average RCF value calculated for different injection volumes was used as the final result, and its relative standard deviation (RSD) reflects the stability of the calculation. To ensure accuracy, all RCF values ​​were uniformly retained to four decimal places. Ginsenosides G-Rk3, G-Rh4, and 20 ( SThe average RCFs of G-Rg3 were 0.5768, 0.4690 and 1.0924, respectively, with a maximum RSD of 1.1%, which is significantly lower than the threshold of 2.0%, confirming that the calculated RCFs have robust stability.

[0069] ③QAMS calculation of Panax notoginseng saponin content

[0070] First, according to ginsenoside 20 ( R The standard curve of 20-Rg3 was used to determine the content of 20-Rg3 in different batches of processed Panax notoginseng. R The content of ginsenoside 20 (Rg3) in different batches of processed Panax notoginseng was determined by QAMS. R The content of )-Rg3.

[0071] According to formula (2), the ginsenosides Rk3, Rh4 and 20 in processed Panax notoginseng were calculated. S The concentration of )-Rg3.

[0072] (2)

[0073] In formula (2), A s and C s (mg / mL) is the internal reference substance ginsenoside 20 ( R Peak area and concentration of )-Rg3, A i and C i (mg / mL) represents the analyte components (ginsenosides Rk3, Rh4, 20) in the processed Panax notoginseng sample. S The peak area and concentration of )-Rg3).

[0074] Finally, the analytes (ginsenosides Rk3, Rh4, 20) in the processed Panax notoginseng sample were calculated according to formula (3). S The content of )-Rg3).

[0075] (3)

[0076] In formula (3), R i (mg / g) represents the analyte components (ginsenosides Rk3, Rh4, 20) in the processed Panax notoginseng sample. S The content of )-Rg3), m (g) is the weight of cooked Panax notoginseng weighed when preparing the sample solution, which is 0.6000g in this invention, and V is the constant volume of the sample solution, which is 50mL in this invention.

[0077] 6. Durability check

[0078] Flow rate variations (0.9, 1.0, and 1.1 mL / min), detection wavelengths (202, 203, and 204 nm), HPLC systems (ThermoFisher Ultimate 3000, Shimadzu LC-20AD, Agilent 1260 Infinity), and columns [Agilent ZORBAX-C18 (4.6 × 250 mm, 5 μm), Shimadzu VP-ODS (4.6 × 250 mm, 5 μm), Inertsil ODS-3C18 (4.6 × 250 mm, 5 μm)] were used to evaluate the efficacy of different concentrations of ginsenosides G-Rk3, G-Rh4, and 20 (G-Rh4) in ginsenosides detected by QAMS. S The effect of the relative correction factor (RCF) of G-Rg3 was systematically reviewed.

[0079] Experimental results are shown in Table 6.

[0080] Table 6. Relative correction factors of saponins under different conditions

[0081] As shown in Table 6, the RCF exhibits good stability under all conditions, with RSDs of flow rate variation <1.0%, wavelength adjustment <1.3%, and system / column variation <2.0%. All deviations are within acceptable limits (5.0%), confirming the robustness of this method for routine application in different laboratory environments.

[0082] 7. Batch variation detection

[0083] The moisture content of three batches (S20230401, S20231113, and S20240201) of processed Panax notoginseng extract was determined according to the drying loss method specified in Part 0832 of the Chinese Pharmacopoeia (2020 Edition). A validated QAMS (Quantitative Analysis of Multi-Components by Single Marker) was used to quantitatively analyze four target sites (G-Rk3, G-Rh4, 20(S)G-Rg3, and 20(R)G-Rg3) in the three batches of processed Panax notoginseng extract.

[0084] Experimental results are shown in Table 7.

[0085] Table 7. Content of four saponins in Panax notoginseng extract.

[0086] Table 7 shows that the methods for quantitative analysis of SPN extract batches (S20230401, S20231113, S20240201) showed good consistency, with the moisture content remaining stable at 2.11±0.06%. Based on dry weight, the four rare ginsenosides exhibited good batch-to-batch reproducibility: G-Rk3: 7.95±0.05% (RSD=0.69%); G-Rh4: 27.78±0.27% (RSD=0.96%); 20 ( S ) G-Rg3: 1.64±0.01% (RSD=0.93%); 20 ( R )G-Rg3: 3.91±0.04% (RSD=1.21%). 20( R The variability of G-Rg3 content (RSD = 1.21%) meets the ICH Q3B acceptable standard for process-related impurities (<5%), while moisture control (average 2.11%) meets the pharmacopoeia standard (USP < 1231). Notably, all labeled RSD values ​​are < 1.5%, demonstrating strong scalability from laboratory scale to optimized pilot-scale production.

[0087] As shown in the above examples and experimental cases, the present invention weighs, grinds, and sieves purified Panax notoginseng roots to obtain particles (2-4 mm). The particles are pretreated by soaking in pure water (100% w / w) for 2 h, and then steamed and dried in a layered tray. The yield of target ginsenosides is improved through a dual mechanism: physical pretreatment: water soaking softens the tissue structure, enhances cell wall permeability, and promotes efficient heat penetration; thermochemical optimization: the target of steaming at 120℃ is the activation energy (Ea≈58.3 kJ / mol) of deglycosylation of protopanaxadiol-type ginsenosides (such as Rb1→Rg3 / Rh2), while 5 h of steaming maximizes the accumulation of rare ginsenosides (such as G-Rk3 and G-Rh4) by balancing conversion kinetics and thermal degradation, and the total saponin content reaches 35.4777 mg / g. The "hydration-controlled thermal conversion" strategy effectively solves the batch variation problem, and the total saponin content achieves consistency between batches.

[0088] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing processed Panax notoginseng, characterized in that, Includes the following steps: 1) Crush raw Panax notoginseng into powder, mix it with water and soak it to obtain moistened Panax notoginseng; 2) Steam and dry Panax notoginseng to obtain cooked Panax notoginseng; The particle size of the pulverized material is 2-4 mm, and the soaking time is 2 hours.

2. The preparation method according to claim 1, characterized in that, Step 1) The temperature of the water is 24.5~25.5℃, the mass ratio of raw Panax notoginseng to water is 1:1, and the mass of water in the soaked raw Panax notoginseng accounts for 100% of the total mass of the substance.

3. The preparation method according to claim 1, characterized in that, Step 1) Drying before pulverization, the drying temperature is 50~55℃, the drying time is 48~52 h, and the drying is carried out until the moisture content is ≤8%.

4. The preparation method according to claim 1, characterized in that, Step 2) The steaming temperature is 100~130℃, and the steaming time is 3~6h.

5. The preparation method according to claim 1, characterized in that, Step 2) The drying temperature is 58~62℃, the drying time is 4~5h, and the drying is carried out until the moisture content is below 9%.

6. The prepared Panax notoginseng obtained by the preparation method according to any one of claims 1 to 5.

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