Preparation method of rare ginsenoside composition and its use in the preparation of products for improving insomnia and memory disorders

Abstract

This invention belongs to the field of biomedical technology, specifically relating to a method for preparing a rare ginsenoside composition and its use in preparing products that improve insomnia and memory disorders. The rare ginsenoside composition obtained by this invention through a specific preparation method consists of ginsenoside Rk3, ginsenoside Rg5, and ginsenoside Rk2. During the preparation process, efficient conversion is achieved by treating ginseng stems and leaves using a stepwise conversion method of enzymatic hydrolysis and fermentation. High-purity separation and purification are completed by chromatography. This method not only solves the problems of insufficient rationality and compatibility of component combinations in existing technologies, but also improves conversion efficiency and reduces the cost of large-scale production through innovative processes, providing a feasible solution for the widespread application of rare ginsenosides in the fields of biomedicine, functional health products, and cosmetics.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the preparation method of rare ginsenoside compositions and their use in the preparation of products that improve insomnia and memory disorders. Background Technology

[0002] Ginseng, a perennial herbaceous plant belonging to the genus Panax of the Araliaceae family, possesses the effects of tonifying the spleen and lungs, replenishing vital energy, calming the mind, and improving intelligence, making it a top choice among natural medicinal materials. Ginsenosides are the main bioactive substances in ginseng. To date, more than 60 ginsenoside monomers have been isolated and identified, among which five major saponins (ginsenosides Rb1, Rb2, Rc, Re, and Rg1) account for more than 80% of the total saponins; rare ginsenosides (such as Rg3, Rh2, Rk1, Rk2, Rk3, and dehydroprotopanaxadiol derivatives) have become the focus of research and application in recent years due to their significantly superior bioactivity compared to conventional saponins. Numerous studies have demonstrated the pharmacological activities of ginsenoside Re in multiple aspects, including anti-oxidative stress damage, anti-inflammation, anti-allergy, anti-cancer, anti-aging, and improving immunity. Simultaneously, studies have shown that ginsenoside Re has some effects on neuroinflammation and neurodegenerative diseases. Especially in the central nervous system, ginsenoside Re improves brain function through antioxidant and anti-inflammatory effects. However, rare ginsenosides are naturally present in very low amounts in fresh ginseng and must be obtained through specific processes. The current technology system for developing compositions based on rare ginsenosides still has many limitations in terms of the compatibility of the components and industrialization support, making it difficult to fully realize the application value of these active ingredients.

[0003] From the perspective of the compatibility of components in rare ginseng compositions, existing technologies have significant shortcomings. Current research and product development often focus on the combination of rare ginsenosides at the level of "single-component application" or "simple mixing without a clear objective," lacking a systematic consideration of the "rationality of multi-component combinations." On the one hand, existing combinations are not specifically designed around application needs (such as oral antioxidants or topical anti-aging). For example, compositions targeting skin aging may simply mix multiple rare ginsenosides without considering the differences in solubility and stability of different components in the skin environment, resulting in some components failing to function effectively. On the other hand, research on the compatibility between components is insufficient. Some rare ginsenosides, under specific combination ratios, may experience decreased solubility and reduced stability due to chemical structural interactions, affecting the overall quality of the composition.

[0004] From the perspective of industrialization support technologies for rare ginseng compositions, existing conversion and purification processes are ill-suited to the demands of large-scale production. The core prerequisite for composition development is obtaining high-purity, stable-supply single rare ginsenoside raw materials. However, existing conversion processes have significant limitations: traditional processing methods are time-consuming and result in large fluctuations in product content; chemical conversion methods easily generate byproducts, affecting raw material purity; and enzyme-catalyzed conversion methods are costly and have limited efficiency in large-scale conversion, leading to persistently high prices for single rare ginsenoside raw materials, directly restricting the batch preparation of compositions. Simultaneously, while existing separation and purification processes (such as conventional chromatography) can obtain high-purity raw materials, they suffer from high solvent consumption, small single-batch processing volumes, and high equipment energy consumption, further increasing the production costs of raw materials and compositions. This makes them unsuitable for mass-market consumer products, limiting their application to niche, high-end sectors.

[0005] In summary, existing technologies for rare ginsenoside compositions face significant bottlenecks in terms of component compatibility and industrialization support. These bottlenecks not only hinder the realization of the application value of rare ginsenosides but also limit their widespread use in biomedicine, functional health products, and cosmetics. Overcoming these technological bottlenecks and achieving rational component combinations and large-scale production of rare ginseng compositions is of great significance for promoting the high-value utilization of ginseng resources and meeting consumer demand for safe and effective functional products. Summary of the Invention

[0006] To address the aforementioned shortcomings, this invention provides a method for preparing a rare ginsenoside composition and its use in preparing products that improve insomnia and memory disorders. The rare ginsenoside composition obtained through a specific preparation method comprises ginsenoside Rk3, ginsenoside Rg5, and ginsenoside Rk2. The preparation process utilizes a stepwise conversion method involving enzymatic hydrolysis and fermentation to treat dried ginseng stems and leaves, achieving efficient conversion. High-purity separation and purification are achieved through chromatography. This method not only solves the problems of insufficient rationality and compatibility in the composition of existing technologies but also improves conversion efficiency and reduces the cost of large-scale production through innovative processes. This provides a feasible solution for the widespread application of rare ginsenosides in the fields of biomedicine, functional health products, and cosmetics.

[0007] To achieve the above objectives, the present invention adopts the following technical solution: A method for preparing a rare ginsenoside composition includes the following steps: (1) Take dried ginseng stems and leaves, wash them clean, dry them, crush them, and obtain granules; (2) Take the particles from step (1) and perform enzymatic hydrolysis, ultrasonic extraction, and freeze-drying to obtain ginseng extract; (3) The ginseng extract obtained in step (2) was fermented with yeast and lactobacillus respectively to obtain ginseng fermentation products; (4) The ginseng fermentation product obtained in step (3) was separated and purified by C30 semi-preparative high performance liquid chromatography to obtain the rare ginsenoside composition.

[0008] Furthermore, the particle size of the particles mentioned in step (1) above is 100-120 mesh; preferably 100 mesh.

[0009] Furthermore, the enzymatic hydrolysis described in step (2) above is performed using cellulase; the ultrasonic vibration power is 200-400 W, the time is 2-3 h, and the temperature is 60-80℃.

[0010] Preferably, the specific operation of step (2) is as follows: take the particles from step (1) and add 8-15 times the mass of distilled water to obtain a mixed solution; add 0.3%-0.5% of cellulase to the mixed solution, adjust the pH value to 3.5-4.5, and extract under ultrasonic and water bath heating at 60-80 ℃ for 2-3 h under the condition of vibration power of 200-400 W, centrifuge and filter to obtain filtrate, repeat the extraction step 2-3 times, combine the filtrates, freeze dry the filtrate to obtain ginseng extract; Furthermore, the yeast mentioned in step (3) above is wine yeast (preservation number: CGMCC2.3997, purchased by Baosai Biotechnology); the lactobacillus mentioned is Lactobacillus acidophilus (preservation number: CGMCC1.1878, purchased by Baosai Biotechnology).

[0011] The method for preparing wine yeast culture is as follows: Pick a loopful of *Wine yeast* colonies and place them in YPD liquid medium. Place the culture on a shaker and serially dilute the activated culture solution onto plates to obtain individual colonies. Pick a single colony from the plate and inoculate it into YPD liquid medium. Incubate at 28°C with a shaker. When the OD value reaches 1.0, the *Wine yeast* fermentation broth is obtained (the strain is in the logarithmic phase, and the concentration is 1×10⁻⁶). 8 (CFU / mL).

[0012] The method for preparing Lactobacillus acidophilus bacterial suspension is as follows: One loopful of Lactobacillus acidophilus colonies was placed in MRS liquid medium and serially diluted on a plate in a shaker to obtain single colonies. A single colony was then inoculated into MRS liquid medium and incubated at 37°C for anaerobic or aerobic culture. When the OD value reached 1.0, the Lactobacillus acidophilus fermentation broth was obtained (the strain was in the logarithmic phase, with a concentration of 1×10⁻⁶). 8 (CFU / mL).

[0013] Furthermore, the mass-to-volume ratio of the ginseng extract to the wine yeast fermentation liquid is 1:1; the mass-to-volume ratio of the ginseng extract to the Lactobacillus acidophilus fermentation liquid is 1:1.

[0014] Preferably, the specific operation of step (3) above is as follows: (3.1) Add distilled water to the ginseng extract obtained in step (2), then add glucose, maltose, casein and ammonium sulfate, sterilize at 115 ℃ for 10-25 min to obtain raw material mixture 1; add wine yeast fermentation liquid to raw material mixture 1 and culture at 28-32 ℃ with shaking for 36-60 h, with a shaking speed of 160-250 rpm / min to obtain ginseng yeast fermentation liquid; (3.2) Add glucose, sucrose, maltose, casein and ammonium sulfate to the ginseng yeast fermentation broth, adjust the pH to 5.5-7.5, sterilize at 115℃ for 10-25 min to obtain raw material mixture 2; add the Lactobacillus acidophilus fermentation broth to the raw material mixture 2 and culture anaerobic or aerobic at 30-37℃ for 36-60 h to obtain ginseng Lactobacillus fermentation broth; (3.3) The fermentation broth of Lactobacillus ginseng obtained in step (3.2) is filtered through diatomaceous earth, the supernatant is collected, and sterilized at 115℃ for 10-25 min to obtain the ginseng fermentation product.

[0015] The mass-to-volume ratio of ginseng extract and distilled water in step (3.1) above is 1g:50mL.

[0016] The mass ratio of the ginseng extract to the mixture of glucose, maltose, casein and ammonium sulfate in step (3.1) above is 1:0.5-1; the concentrations of glucose, maltose, casein and ammonium sulfate are 0.01-0.02 g / mL, 0.01-0.02 g / mL, 0.01-0.02 g / mL and 0.01-0.02 g / mL, respectively.

[0017] The mass ratio of the ginseng extract to the mixture of glucose, maltose, casein and ammonium sulfate in step (3.2) above is 1:0.5-1; the concentrations of glucose, maltose, casein and ammonium sulfate are 0.01-0.02 g / mL, 0.01-0.02 g / mL, 0.01-0.02 g / mL and 0.01-0.02 g / mL, respectively.

[0018] The specific steps for separation and purification in step (4) above are as follows: Chromatographic column: C30 column (250 mm × 21.2 mm, 5 μm) was used. Column temperature: 28 ℃; Mobile phase A is water, and mobile phase B is 85% acetonitrile; The isocratic elution conditions were 55% B - 45% A; Flow rate: 5 mL / min for 0-30 min, and 2.5 mL / min after 30 min; The detection wavelength is 203 nm.

[0019] Ginsenoside Rk3 was collected at 34-38 min, ginsenoside Rg5 at 54-59 min, and ginsenoside Rk2 at 85-90 min.

[0020] The present invention also provides a rare ginsenoside composition prepared by the above preparation method, wherein the rare ginsenoside composition is composed of ginsenoside Rk3, ginsenoside Rg5 and ginsenoside Rk2 in a mass ratio of 1-3:4-6:1-3.

[0021] The present invention also provides the use of the rare ginsenoside composition prepared by the above preparation method in the preparation of products that improve insomnia and memory disorders.

[0022] The products mentioned include pharmaceuticals or health supplements.

[0023] A medicament for improving insomnia and memory disorders, the medicament comprising the above-mentioned rare ginsenoside composition and pharmaceutically acceptable excipients.

[0024] A health supplement for improving insomnia and memory disorders, the health supplement comprising the above-mentioned rare ginsenoside composition and excipients acceptable in the health supplement.

[0025] The beneficial effects of this invention are as follows: (1) This invention utilizes enzymatic extraction-biofermentation synergistic processing, combined with semi-preparative high performance liquid chromatography purification, to obtain high-purity, batch-stable ginsenosides Rk3, Rg5 and Rk2, solving the problems of long process cycle, large product fluctuation and low purification efficiency in existing processes.

[0026] (2) The present invention has been verified by animal experiments. The composition can slow down the weight loss trend of insomnia mice, reduce the spontaneous activity time and the number of times insomnia mice stand up, increase the level of 5-HT and GABA in brain tissue, and decrease the level of Glu in brain.

[0027] (3) This experiment has been verified by animal experiments. The composition can reduce the number of errors in the platform test and prolong the latency period in mice with memory impairment; significantly increase the DI value in the new object recognition test and effectively enhance cognitive memory ability; significantly increase the number of platform crossings in the water maze test and reduce the latency period and effectively improve spatial memory ability. Attached Figure Description

[0028] Figure 1 The changes in body weight of mice in each group in Example 1 are shown. Compared with the control group, ### p <0.001; compared with the model group, p <0.001, p <0.01.

[0029] Figure 2 This refers to the effect of RGCs on spontaneous activity in mice in Example 1, where, compared to the control group, ### p <0.001; compared with the model group, p <0.05, p <0.01.

[0030] Figure 3 This refers to the effect of RGC on neurotransmitter levels in mouse brain tissue in Example 1, where, compared to the control group, ### p <0.001; compared with the model group, p <0.05, p <0.01, p< 0.001.

[0031] Figure 4 The latency (A) and number of errors (B) of mice in each group during the step-down experiment in Example 1 are shown below, compared to the control group. ### p <0.001; compared to the model group, p <0.05, p <0.01, p <0.001.

[0032] Figure 5 This is the new object recognition result for Example 1, where, compared to the blank group, ### p <0.001; compared to the model group, p <0.01, p <0.001.

[0033] Figure 6 The results of the water maze in Example 1 are shown (A: number of platform crossings; B: latency period). Compared to the control group, ### p <0.001; compared to the model group, p <0.05, p <0.01, p <0.001.

[0034] Figure 7 The high-performance liquid chromatogram of ginsenosides prepared in the example is shown below. Figure 8 The chromatographic peaks of ginsenoside Rk3 sample (A) and ginsenoside Rk3 standard (B) prepared in Example 1 are shown. Figure 9 The chromatographic peaks of ginsenoside Rg5 sample (A) and ginsenoside Rg5 standard (B) prepared in Example 1 are shown.

[0035] Figure 10 The chromatographic peaks of ginsenoside Rk2 sample (A) and ginsenoside Rk2 standard (B) prepared in Example 1 are shown. Detailed Implementation The present invention will be further clearly and completely illustrated below through embodiments. These embodiments are only some examples of the present invention and are not intended to limit the present invention, but are only for illustrating the present invention. Unless otherwise specified, the experimental methods used in the following embodiments are all conventional experiments, and the materials and reagents used in the following embodiments are commercially available unless otherwise specified.

[0036] The experimental materials and instruments are shown in Table 1.

[0037] Table 1 Main Reagents and Instruments

[0038] Basic Example 1: The preparation method of wine yeast culture is as follows: Pick a loopful of *Wine yeast* colonies and place them in YPD liquid medium. Place the culture on a shaker and serially dilute the activated culture solution onto plates to obtain individual colonies. Pick a single colony from the plate and inoculate it into YPD liquid medium. Incubate at 28°C with a shaker. When the OD value reaches 1.0, the *Wine yeast* fermentation broth is obtained (the strain is in the logarithmic phase, and the concentration is 1×10⁻⁶). 8 (CFU / mL).

[0039] Basic Example 2: The preparation method of Lactobacillus acidophilus bacterial solution is as follows: One loopful of Lactobacillus acidophilus colonies was placed in MRS liquid medium and serially diluted on a plate in a shaker to obtain single colonies. A single colony was then inoculated into MRS liquid medium and incubated at 37°C for anaerobic or aerobic culture. When the OD value reached 1.0, the Lactobacillus acidophilus fermentation broth was obtained (the strain was in the logarithmic phase, with a concentration of 1×10⁻⁶). 8 (CFU / mL).

[0040] Example 1: A method for preparing a rare ginsenoside composition Includes the following steps: (1) Take 100g of dried ginseng stems and leaves, wash them clean and dry them, crush them and pass them through a 100-mesh sieve to obtain granules; (2) Take the particles from step (1) and add 8 times the mass of distilled water to obtain a mixed solution; add 0.3% of cellulase to the mixed solution, adjust the pH to 4, and extract by ultrasonication and water bath heating at 60°C for 3 h under the condition of vibration power of 300 W. Centrifuge and filter to obtain filtrate. Repeat the extraction step 3 times, combine the filtrates, freeze dry the filtrate to obtain 50g of ginseng extract; (3) The ginseng extract obtained in step (2) was fermented with yeast and lactobacillus respectively to obtain ginseng fermentation products; Specifically: (3.1) Add 50 times (w / v) of distilled water to 50g of ginseng extract obtained in step (2), and then add 50g of a mixture of glucose, maltose, casein and ammonium sulfate to make the concentrations 0.02 g / mL glucose, 0.02 g / mL maltose, 0.02 g / mL casein and 0.02 g / mL ammonium sulfate respectively. Sterilize at 115 ℃ for 10-25 min to obtain raw material mixture 1; add wine yeast fermentation liquid with a mass-volume ratio of 1:1 to ginseng extract to raw material mixture 1 and shake and culture at 30℃ for 48h with a shaking speed of 200 rpm / min to obtain ginseng yeast fermentation liquid; (3.2) Add 50g of a mixture of glucose, maltose, casein and ammonium sulfate to 2500g of ginseng yeast fermentation broth to make the concentrations 0.02 g / mL glucose, 0.02 g / mL maltose, 0.02 g / mL casein and 0.02 g / mL ammonium sulfate respectively. Adjust the pH to 7 and sterilize at 115℃ for 20 min to obtain raw material mixture 2. Add Lactobacillus acidophilus fermentation broth with a mass-volume ratio of 1:1 to ginseng extract to raw material mixture 2 and culture at 35℃ for anaerobic or aerobic conditions for 56 h to obtain ginseng Lactobacillus fermentation broth. (3.3) The fermentation broth of Lactobacillus ginseng obtained in step (3.2) is filtered through diatomaceous earth, the supernatant is collected, and sterilized at 115℃ for 20 min to obtain the ginseng fermentation product; (4) The ginseng fermentation product obtained in step (3) was separated and purified by C30 semi-preparative high performance liquid chromatography to obtain the rare ginsenoside composition. Specifically: Chromatographic column: C30 column (250 mm × 21.2 mm, 5 μm) was used. Column temperature: 28 ℃; Mobile phase A is water, and mobile phase B is 85% acetonitrile; The isocratic elution conditions were 55% B - 45% A; Flow rate: 5 mL / min for 0-30 min, and 2.5 mL / min after 30 min; The detection wavelength is 203 nm.

[0041] Ginsenoside Rk3 was collected at 34-38 min, ginsenoside Rg5 at 54-59 min, and ginsenoside Rk2 at 85-90 min. (See attached text) Figure 7 .

[0042] Identification revealed that the obtained rare ginsenoside composition was a mixture of ginsenoside Rk3, ginsenoside Rg5, and ginsenoside Rk2 in a mass ratio of 2:4:1.

[0043] The prepared samples were analyzed to confirm that the purified samples were ginsenoside Rk3, ginsenoside Rg5, and ginsenoside Rk2. Purchased standards and the prepared ginsenosides Rk3, Rg5, and Rk2 were analyzed by high-performance liquid chromatography (HPLC) using the following method: Waters C18 column (4.6 mm × 250 mm, 5 μm); The mobile phase is 10% acetonitrile aqueous solution (A) - 80% acetonitrile solution (B); Gradient elution (0-14 min, 86%→84% A; 14-24 min, 84%→83% A; 24-36 min, 83%→81% A; 36-60 min, 81%→57% A; 60-82 min, 57%→15% A; 82-98 min, 15%→0% A; 98-110 min; 0%→100% A); Column temperature 30℃; The flow rate was 1.3 mL / min from 0 to 60 min and 1.0 mL / min from 60 to 109.5 min. The detection wavelength is 203 nm.

[0044] Rk3 peak elution time: 69.202; Rg5 elution time: 81.034; Rk2 peak time: 93.409.

[0045] For detailed analysis of the chromatogram, please refer to... Figure 8-10 Comparison revealed that the peak positions of ginsenosides Rk3, Rg5, and Rk2 were the same as those of the purchased ginsenoside Rk3, Rg5, and Rk2 standards, thus proving that the present invention successfully isolated and purified ginsenosides Rk3, Rg5, and Rk2.

[0046] Example 2: A method for preparing a rare ginsenoside composition Includes the following steps: (1) Take 100g of dried ginseng stems and leaves, wash them clean and dry them, crush them and pass them through a 100-mesh sieve to obtain granules; (2) Take the particles from step (1) and add 10 times the mass of distilled water to obtain a mixed solution; add 0.3% of cellulase to the mixed solution, adjust the pH to 4, and extract by ultrasonication and water bath heating at 80 ℃ for 2 h under the condition of vibration power of 300 W. Centrifuge and filter to obtain filtrate. Repeat the extraction step 3 times, combine the filtrates, freeze dry the filtrate to obtain 50g of ginseng extract; (3) The ginseng extract obtained in step (2) was fermented with yeast and lactobacillus respectively to obtain ginseng fermentation products; Specifically: (3.1) Add 50 times (w / v) of distilled water to 50g of ginseng extract obtained in step (2), and then add 50g of a mixture of glucose, maltose, casein and ammonium sulfate to make the concentrations 0.02 g / mL glucose, 0.02 g / mL maltose, 0.02 g / mL casein and 0.02 g / mL ammonium sulfate respectively. Sterilize at 115 ℃ for 10-25 min to obtain raw material mixture 1; add wine yeast fermentation liquid with a mass-volume ratio of 1:1 to ginseng extract to raw material mixture 1 and shake and culture at 30℃ for 48h with a shaking speed of 200 rpm / min to obtain ginseng yeast fermentation liquid; (3.2) Add 50g of a mixture of glucose, maltose, casein and ammonium sulfate to 2500g of ginseng yeast fermentation broth to make the concentrations 0.02 g / mL glucose, 0.02 g / mL maltose, 0.02 g / mL casein and 0.02 g / mL ammonium sulfate respectively. Adjust the pH to 7 and sterilize at 115℃ for 20 min to obtain raw material mixture 2. Add Lactobacillus acidophilus fermentation broth with a mass-volume ratio of 1:1 to ginseng extract to raw material mixture 2 and culture at 35℃ for anaerobic or aerobic conditions for 56 h to obtain ginseng Lactobacillus fermentation broth. (3.3) The fermentation broth of Lactobacillus ginseng obtained in step (3.2) is filtered through diatomaceous earth, the supernatant is collected, and sterilized at 115℃ for 20 min to obtain the ginseng fermentation product; (4) The ginseng fermentation product obtained in step (3) was separated and purified by C30 semi-preparative high performance liquid chromatography to obtain the rare ginsenoside composition. Specifically: Chromatographic column: C30 column (250 mm × 21.2 mm, 5 μm) was used. Column temperature: 28 ℃; Mobile phase A is water, and mobile phase B is 85% acetonitrile; The isocratic elution conditions were 55% B - 45% A; Flow rate: 5 mL / min for 0-30 min, and 2.5 mL / min after 30 min; The detection wavelength is 203 nm.

[0047] Ginsenoside Rk3 was collected at 34-38 min, ginsenoside Rg5 at 54-59 min, and ginsenoside Rk2 at 85-90 min.

[0048] Identification revealed that the obtained rare ginsenoside composition was a mixture of ginsenoside Rk3, ginsenoside Rg5, and ginsenoside Rk2 in a mass ratio of 1:3:1.

[0049] Example 3: A method for preparing a rare ginsenoside composition Includes the following steps: (1) Take 100g of dried ginseng stems and leaves, wash them clean and dry them, crush them and pass them through a 100-mesh sieve to obtain granules; (2) Take the particles from step (1) and add 10 times the amount of distilled water to obtain a mixed solution; add 0.5% of cellulase to the mixed solution, adjust the pH to 4, and extract by ultrasonication and water bath heating at 80 ℃ for 3 h under the condition of vibration power of 400 W. Centrifuge and filter to obtain filtrate. Repeat the extraction step 3 times, combine the filtrates, freeze dry the filtrate to obtain 60g of ginseng extract; (3) The ginseng extract obtained in step (2) was fermented with yeast and lactobacillus respectively to obtain ginseng fermentation products; Specifically: (3.1) Add 50 times (w / v) of distilled water to 60g of ginseng extract obtained in step (2), and then add 60g of a mixture of glucose, maltose, casein and ammonium sulfate to make the concentrations 0.02 g / mL glucose, 0.02 g / mL maltose, 0.02 g / mL casein and 0.02 g / mL ammonium sulfate respectively. Sterilize at 115 ℃ for 10-25 min to obtain raw material mixture 1; add wine yeast fermentation liquid with a mass-volume ratio of 1:1 to ginseng extract to raw material mixture 1 and shake at 30℃ for 48h with a shaking speed of 200 rpm / min to obtain ginseng yeast fermentation liquid; (3.2) Add 60g of a mixture of glucose, maltose, casein and ammonium sulfate to 3000g of ginseng yeast fermentation broth to make the concentrations 0.02 g / mL glucose, 0.02 g / mL maltose, 0.02 g / mL casein and 0.02 g / mL ammonium sulfate respectively, adjust the pH to 7, sterilize at 115℃ for 20 min to obtain raw material mixture 2; add Lactobacillus acidophilus fermentation broth with a mass-volume ratio of 1:1 to ginseng extract to raw material mixture 2 and culture at 35℃ anaerobic or aerobic for 56 h to obtain ginseng Lactobacillus fermentation broth; (3.3) The fermentation broth of Lactobacillus ginseng obtained in step (3.2) is filtered through diatomaceous earth, the supernatant is collected, and sterilized at 115℃ for 20 min to obtain the ginseng fermentation product; (4) The ginseng fermentation product obtained in step (3) was separated and purified by C30 semi-preparative high performance liquid chromatography to obtain the rare ginsenoside composition. Specifically: Chromatographic column: C30 column (250 mm × 21.2 mm, 5 μm) was used. Column temperature: 28 ℃; Mobile phase A is water, and mobile phase B is 85% acetonitrile; The isocratic elution conditions were 55% B - 45% A; Flow rate: 5 mL / min for 0-30 min, and 2.5 mL / min after 30 min; The detection wavelength is 203 nm.

[0050] Ginsenoside Rk3 was collected at 34-38 min, ginsenoside Rg5 at 54-59 min, and ginsenoside Rk2 at 85-90 min.

[0051] Identification revealed that the obtained rare ginsenoside composition was a mixture of ginsenoside Rk3, ginsenoside Rg5, and ginsenoside Rk2 in a mass ratio of 2:6:1.

[0052] Comparative Example 1: The difference from Example 3 is that yeast fermentation is not performed, i.e., step (3.1) is omitted, specifically: (3) The ginseng extract obtained in step (2) is fermented with Lactobacillus to obtain ginseng fermentation product; Specifically: (3.1) Add 50 times (w / v) of distilled water to 60g of ginseng extract obtained in step (2), and then add 60g of a mixture of glucose, maltose, casein and ammonium sulfate to make the concentrations 0.02 g / mL glucose, 0.02 g / mL maltose, 0.02 g / mL casein and 0.02 g / mL ammonium sulfate respectively. Adjust the pH to 7 and sterilize at 115℃ for 20 min to obtain raw material mixture 1. Add the Lactobacillus acidophilus fermentation broth with a mass-volume ratio of 1:1 to the raw material mixture 2 and culture it anaerobically or aerobically at 35℃ for 56 h to obtain Lactobacillus ginseng fermentation broth. (3.2) The fermentation broth of Lactobacillus ginseng obtained in step (3.1) is filtered through diatomaceous earth, the supernatant is collected, and sterilized at 115℃ for 20 min to obtain the ginseng fermentation product.

[0053] Everything else is the same as in Example 3.

[0054] Comparative Example 2: The difference from Example 3 is that lactobacillus fermentation is not performed, i.e., step (3.2) is omitted, specifically: (3) The ginseng extract obtained in step (2) is fermented with yeast to obtain ginseng fermentation product; Specifically: (3.1) Add 50 times (w / v) of distilled water to 60g of ginseng extract obtained in step (2), and then add 60g of a mixture of glucose, maltose, casein and ammonium sulfate to make the concentrations 0.02 g / mL glucose, 0.02 g / mL maltose, 0.02 g / mL casein and 0.02 g / mL ammonium sulfate, respectively. Sterilize at 115 ℃ for 20 min to obtain raw material mixture 1; add wine yeast fermentation liquid with a mass-volume ratio of 1:1 to ginseng extract to raw material mixture 1 and shake at 30℃ for 48 h with a shaking speed of 200 rpm / min to obtain ginseng yeast fermentation liquid; (3.2) The ginseng yeast fermentation broth obtained in step (3.1) is filtered through diatomaceous earth, the supernatant is collected, and sterilized at 115℃ for 20 min to obtain the ginseng fermentation product. Everything else is the same as in Example 3.

[0055] Comparative Example 3: The difference from Example 1 is that steps (3.1) and (3.2) are reversed, specifically: (3) The ginseng extract obtained in step (2) was fermented with Lactobacillus and yeast respectively to obtain ginseng fermentation products; Specifically: (3.1) Add 50 times (w / v) of distilled water to 60g of ginseng extract obtained in step (2), and then add 60g of a mixture of glucose, maltose, casein and ammonium sulfate to make the concentrations 0.02 g / mL glucose, 0.02 g / mL maltose, 0.02 g / mL casein and 0.02 g / mL ammonium sulfate, respectively. Adjust the pH to 7 and sterilize at 115℃ for 20 min to obtain raw material mixture 1. Add Lactobacillus acidophilus fermentation broth with a mass-volume ratio of 1:1 to ginseng extract to raw material mixture 1 and culture at 35℃ for anaerobic or aerobic conditions for 56 h to obtain ginseng Lactobacillus fermentation broth. (3.2) Add 60g of a mixture of glucose, maltose, casein and ammonium sulfate to 3000g of ginseng yeast fermentation broth, so that the concentrations are 0.02 g / mL glucose, 0.02 g / mL maltose, 0.02 g / mL casein and 0.02 g / mL ammonium sulfate, respectively. Sterilize at 115 ℃ for 20 min to obtain raw material mixture 2. Add wine yeast fermentation broth with a mass-volume ratio of 1:1 to ginseng extract to raw material mixture 2 and culture at 30℃ with shaking for 48 h at a shaking speed of 200 rpm / min to obtain ginseng yeast fermentation broth. (3.3) The fermentation broth of Lactobacillus ginseng obtained in step (3.2) is filtered through diatomaceous earth, the supernatant is collected, and sterilized at 115℃ for 20 min to obtain the ginseng fermentation product; Everything else is the same as in Example 3.

[0056] Effect Experiment: Example 1: Sedative and hypnotic effects of the composition 1.1 Experimental Methods 1.1.1 Experimental Materials 1.1.1.1 Instruments EPOCH microplate reader (Bio Tek); TGL-16aR ultracentrifuge (Shanghai Anting Scientific Instrument Factory); SHB-IIIA circulating water multi-purpose vacuum pump (Shanghai Yukang Scientific and Educational Instrument Equipment Co., Ltd.); R201D constant temperature water bath (Shanghai Yukang Scientific and Educational Instrument Equipment Co., Ltd.); RE-2000A rotary evaporator (Shanghai Xiande Experimental Instrument Co., Ltd.); DLSB-5 / 20 low temperature coolant circulating pump (Shanghai Yukang Scientific and Educational Instrument Equipment Co., Ltd.); PTX-FA21OS analytical balance (Fujian Huazhi Electronic Technology Co., Ltd.); DZF-6020 vacuum drying oven (Shanghai Boxun Industrial Co., Ltd.); KQ-250B ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.).

[0057] 1.1.1.2 Laboratory Animals SPF-grade adult Kunming mice, half male and half female, weighing 18-22 g, were purchased from Changchun Yisi Laboratory Animal Technology Co., Ltd. The animals were housed under standard laboratory conditions (temperature 25±2℃, 12h light / 12h dark cycle, relative humidity 60±5%), with no restrictions on water or food intake. Animal handling was performed in accordance with the "Guide for the care and use of laboratory animals".

[0058] 1.1.2 Animal grouping, drug administration, and construction of insomnia model After one week of acclimatization, mice were randomly divided into 6 groups (n=6) according to their body weight, including: blank group, model group, positive control group (Diazepam, 6.5 mg / kg) (batch number: 220202, purchased from Southwest Pharmaceutical), Example 1 group (20 mg / kg), Example 2 group (20 mg / kg), Example 3 group (20 mg / kg), Comparative Example 1 group (20 mg / kg), Comparative Example 2 group (20 mg / kg), and Comparative Example 3 group (20 mg / kg).

[0059] First, prophylactic administration was carried out. Administration was performed daily around 9:00 AM. The RGC administration group received the drug via gavage, the positive control group received the drug via intraperitoneal injection, and the control and model groups received an equal volume of physiological saline via gavage. The administration volume was 10 mL / kg for 7 consecutive days.

[0060] Next, an insomnia model was established. Except for the control group, which received an equal volume of physiological saline intraperitoneally, all other groups received an intraperitoneal injection of p-chlorophenylalanine saline solution (catalog number: S93084, purchased from Shanghai Yuanye Biotechnology Co., Ltd.) (350 mg / kg) once daily for 4 days to establish the insomnia model. Compared with the control group, mice exhibited restlessness, increased excitability, increased aggression, continuous activity, loss of diurnal rhythm, decreased appetite, increased urine and fecal production, dull and disheveled fur, and weight loss, indicating a preliminary successful model establishment.

[0061] Finally, therapeutic drug administration was performed. After successful modeling, drugs were administered daily at approximately 9:00 AM. The control and comparative groups were administered drugs by gavage, the positive control group was administered drugs by intraperitoneal injection, and the blank control and model groups were administered an equal volume of physiological saline by gavage. The drug administration volume was 10 mL / kg for 7 consecutive days.

[0062] 1.1.3 Effects on behavioral changes, body weight, and spontaneous activity in mice Observe the behavioral changes of mice daily, weigh and record the weight of the mice, and compare the weight changes of mice at different drug administration stages. On the 6th day of drug administration, 60 minutes after drug administration, each mouse was placed in a cage alone. After 3 minutes of acclimatization activities, the spontaneous activity time and the number of times the mice stood up (the number of times the forelimbs were raised) were recorded continuously for 5 minutes.

[0063] 1.1.4 Effects on the levels of neurotransmitters 5-HT, GABA, and Glu in mouse brain tissue Sixty minutes after the last administration, mice in each group were euthanized by decapitation. The brain tissue of the mice was quickly dissected and removed. After rinsing with pre-cooled PBS buffer and drying, the weight was recorded. The same weight of brain tissue was weighed and chopped. The tissue was then placed in an ice-cold tissue homogenizer, and nine times the amount of pre-cooled PBS buffer was added. The homogenizer was homogenized on ice and centrifuged at 12,000 rpm for 10 minutes. The supernatant was collected to obtain the brain tissue homogenate sample.

[0064] The contents of 5-HT, GABA and Glu in mouse brain tissue were determined strictly according to the instructions of the ELISA kits (5-HT kit No.: FY2443-A; GABA kit No.: FY2442-A; Glu kit No.: FY30031-A, purchased from Jiangsu Feiya Biotechnology Co., Ltd.).

[0065] 1.1.5 Data Statistics and Analysis All measurement data are expressed as mean ± standard deviation (SD). Data processing was performed using GraphPad Prism 10.1.2 statistical analysis software. One-way analysis of variance (ANOVA) was used to test whether there were significant differences between groups. A p < 0.05 was used to indicate a significant difference and statistical significance. The test results are shown in Table 2 below.

[0066] 1.2 Experimental Results 1.2.1 Effects of RGCs on behavioral phenomena and body weight changes in mice Behavioral observations of mice revealed that before modeling, the mice slept normally, were in good spirits, and had smooth, shiny fur. After modeling, compared with the control group, the mice in the other modeling groups had dull, disheveled fur, exhibited restlessness, increased aggression, loss of circadian rhythm, decreased appetite, and increased urination and defecation, indicating preliminary success in modeling. After drug administration, the mental state of the mice in each group gradually stabilized, and their sleep became more regular, suggesting that RGC has a certain ameliorative effect on insomnia in mice.

[0067] Representative body weights were selected to plot weight change curves, specifically day 1 (starting weight), day 7 (last day of prophylactic administration), day 11 (last day of modeling), and day 18 (last day of therapeutic administration). Weight changes are shown in the figure. Figure 1 The results showed that on day 11, the fourth day after intraperitoneal injection of pCPA, the body weight of mice in all groups except the control group decreased significantly, indicating that the model had been successfully established. On day 18, after therapeutic administration, compared with the model group, the body weight of mice in the positive control group, Example 2, and Example 3 all increased significantly (p<0.05, p<0.001), with the positive control group showing the most significant increase. These findings suggest that RGC has a certain ameliorative effect on insomnia in mice.

[0068] Table 2 Effect of RGC on mouse body weight on day 18

[0069] Note: Compared with the blank group, ### p<0.001; compared with the model group, p<0.05, p<0.001.

[0070] 1.2.2 Effects on spontaneous activity time and number of standing times in mice Insomnia is a complex physiological process involving multiple systems. The functional state of the central nervous system can be externally manifested in the spontaneous activity of mice; increased activity frequency indicates central nervous system excitation, while decreased frequency indicates central nervous system inhibition. Therefore, this indicator can be used to assess the state of the central nervous system. This experiment recorded and analyzed the spontaneous activity time and number of standing movements within 5 minutes in each group of mice within 1 hour after drug administration (see Table 3). Figure 2 .

[0071] The results showed that, compared with the control group, the activity time and number of standing times of the model group mice were significantly increased (p<0.001), indicating that the mouse insomnia model was successfully established. Regarding spontaneous activity time, all mice in the examples showed a significant decrease compared with the model group (p<0.001), while mice in Example 3 showed a significant increase compared with all control group mice (p<0.01, p<0.01). Regarding the number of standing times, mice in Example 2 and Example 3 showed a significant decrease compared with the model group (p<0.01, p<0.001), while mice in Example 3 showed a significant decrease compared with all control group mice (p<0.01, p<0.05). This indicates that RGC has a certain sedative effect and can inhibit spontaneous activity in mice, but its effect is not as good as that of the positive control drug diazepam.

[0072] Table 3 Effects of RGCs on spontaneous activity in mice

[0073] Note: Compared with the blank group, ### p<0.001; compared with the model group, p<0.01, p<0.001, compared with Example 3, △ p<0.01, △△ p<0.01.

[0074] 1.2.3 Effects on the levels of neurotransmitters 5-HT, GABA, and Glu in mouse brain tissue Insomnia is a neuroregulatory process involving numerous neurotransmitters. Disruptions in the balance between excitation and inhibition in the central nervous system can cause changes in the levels of neurotransmitters in the brain. Among these, 5-HT, GABA, and Glu are crucial neurotransmitters closely related to the occurrence and development of insomnia. This study measured the effects of RGC on the levels of the neurotransmitters 5-HT, GABA, and Glu in mouse brain tissue. The results are shown in Table 4. Figure 3 .

[0075] The results showed that, compared with the control group, the levels of 5-HT and GABA in the brain tissue of mice in the model group were significantly reduced (p<0.001), while the level of Glu was significantly increased (p<0.001), indicating that the mouse insomnia model was successfully established. For 5-HT, compared with the model group, the positive drug and all examples significantly increased its level in mouse brain tissue (p<0.001, p<0.01, p<0.01, p<0.01), and Example 3 significantly increased it compared with all comparative mice (p<0.01, p<0.01, p<0.05). For GABA, compared with the model group, the positive drug group and all examples significantly increased its expression in the brain (p<0.001, p<0.01, p<0.01, p<0.001), and Example 3 significantly increased it compared with all comparative mice (p<0.05). For Glu, compared with the model group, the positive drug group and all examples significantly reduced its content in the brain (p<0.001, p<0.01), and Example 3 significantly increased it compared with all comparative mice (p<0.05). The results above indicate that RGC can achieve a sedative and sleep-inducing effect by regulating the levels of neurotransmitters 5-HT, GABA, and Glu in mouse brain tissue.

[0076] Table 4. Effects of RGC on neurotransmitter levels in mouse brain tissue.

[0077] Note: Compared with the blank group, ### p<0.001; compared with the model group, p<0.01, p<0.001; compared with Example 3, △ p<0.05, △△ p<0.01.

[0078] In summary, this invention evaluated the sedative and hypnotic effects of erythropoietin (RGC), expanding the drug applications of RGC. A mouse insomnia model was established using intraperitoneal injection of p-chlorophenylalanine (pCPA). The results showed that RGC could alleviate pCPA-induced behavioral changes in insomnia mice, such as loss of circadian rhythm and restlessness, increase mouse body weight, and inhibit spontaneous activity. It also increased the levels of neurotransmitters 5-HT and GABA in the brain tissue of insomnia mice while decreasing the level of Glu, suggesting that RGC has a certain sedative and hypnotic effect, and its mechanism of action may be related to the regulation of neurotransmitter levels in the brain.

[0079] Effect Example 2: Effect of the composition on scopolamine-induced memory acquisition impairment in mice 2.1 Experimental Methods 2.1.1 Experimental Materials 2.1.1.1 Materials and Reagents Laboratory animals: Male BALB / c mice (20-22 g), purchased from Changchun Yisi Laboratory Animal Technology Co., Ltd. They were housed in a barrier environment facility of the School of Basic Medical Sciences, Jilin University, at a temperature of 22-24℃ and humidity of 45%-55%, with a 12-hour light-dark cycle. During the acclimatization period, the mice had free access to food and water.

[0080] Reagent: Scopolamine, purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.

[0081] 2.1.1.2 Instruments See Table 5: Table 5 Instruments

[0082] 2.1.2 Animal Grouping After 7 days of acclimatization, the mice were randomly divided into 6 groups (n=6): blank group, model group, positive drug group (70 μg / kg, huperzine A), Example 1 group (80 mg / kg), Example 2 group (80 mg / kg), Example 3 group (80 mg / kg), Comparative Example 1 group (80 mg / kg), Comparative Example 2 group (80 mg / kg), and Comparative Example 3 group (80 mg / kg).

[0083] 2.1.3 Construction of the Memory Impairment Model Each treatment group was administered the corresponding dose of each drug via gavage once daily, with a gavage volume of 10 mL / kg. The control group and model group were administered the same volume of distilled water via gavage daily for 30 consecutive days. Except for the control group, all other groups of mice were injected intraperitoneally with scopolamine (5 mg / kg) 10 minutes before the start of behavioral experiment training to establish a mouse model of memory acquisition impairment. Mice in the control group were injected intraperitoneally with the same volume of physiological saline.

[0084] 2.1.4 Diving Platform Experiment The mice were trained for the jumping platform experiment the day after the last administration. The jumping platform apparatus consisted of a reaction chamber and a recording device. The reaction chamber contained an insulated platform and a copper grid. The mice were placed in the reaction chamber (on and off the platform) for 3 minutes to acclimatize. Then, a 36V AC current was applied. After receiving an electric shock on the copper grid in the reaction chamber, the mice reacted normally and jumped back to the insulated platform to avoid the noxious stimulus. After one training session, the mice were placed on the platform, and the latency (the time from the start of the experiment to the animal's first jump off the insulated platform) and the number of errors within 5 minutes (the number of times the animal jumped off the platform and was shocked within 5 minutes) were recorded. If the latency exceeded 3 minutes, the latency was considered to be 180 seconds.

[0085] 2.1.5 New Object Recognition Experiment The first phase was the adaptation period, lasting 3 days, conducted at fixed times each day. Mice were placed in an experimental chamber (58×43×34cm) and allowed to explore freely for 10 minutes without any additional objects. After each training session, the inner walls of the chamber were first rinsed with distilled water, then thoroughly wiped and disinfected with a 75% ethanol solution. The chamber was then ventilated and dried before the next batch of experiments could begin. All operations were performed under constant temperature (22-24℃), constant humidity (45%-55%), and constant light conditions.

[0086] The second phase is the training period, which takes place the day after the adaptation period. First, two identical objects (wooden blocks) are fixed symmetrically on both sides of the experimental box (approximately 10 cm from the box wall). The mouse is placed in the experimental box and allowed to freely explore the two objects for 10 minutes. After training, the mouse is returned to its cage, and the experimental box is cleaned.

[0087] The third phase is the testing period, conducted 1 hour after the training period ends. One of the objects from the training period is replaced with a new object, and the mouse is then placed back in the experimental box to explore freely for 10 minutes. During this period, the time taken for the mouse to explore the new and old objects is recorded. The discrimination index (DI) is used as the evaluation metric. DI (%) = (exploration time of the new object - exploration time of the old object) / (exploration time of the new object + exploration time of the old object) × 100%. A higher DI value indicates a stronger memory ability in the mouse.

[0088] 2.1.6 Morris Water Maze Experiment The Morris Water Maze (MWM) consists of two parts: a circular water tank and an automated camera and analysis system. The circular water tank is 50 cm high and 120 cm in diameter, divided into four areas. Each area has a 10 cm diameter platform. The walls of the four areas are decorated with different patterns. The water level is 20 cm high, and the water temperature is 19-22 ℃.

[0089] The first five days were dedicated to a navigational training experiment, with the platform placed 2 cm underwater. Mice were trained in four different areas of the tank, each session lasting 60 seconds. The mice were positioned with their faces towards a marker on the tank wall, held for 10 seconds, and then placed into the water from that location, simultaneously activating the timing button on the analysis system. If a mouse failed to locate the platform within 60 seconds, it was guided to swim to the platform and remain there for 20 seconds to reinforce its memory of the platform. Each mouse was entered from each of the four quadrants to train its spatial memory. After the experiment, the mice were dried with a towel and returned to their cages.

[0090] On day 6, the space exploration experiment was conducted. The platform was removed, and the mice were released into the water from the quadrant furthest from the original platform location. An automatic recorder recorded the latency period (the time it took for the animal to first find the hidden platform area after being released from the water) and the number of times the animal crossed the platform (the number of times the animal traversed the original platform area after the platform was removed).

[0091] 2.1.7 Data Statistics and Analysis All measurement data are expressed as mean ± standard deviation (SD). Data processing was performed using GraphPad Prism 10.1.2 statistical analysis software. One-way analysis of variance (ANOVA) was used to test whether there were significant differences between groups. A p < 0.05 was used to indicate a significant difference and statistical significance. The test results are shown in Table 6 below.

[0092] 2.2 Experimental Results 2.2.1 Results of the Diving Platform Experiment In the platform jumping test, latency is a crucial indicator for evaluating the positivity of the result; a longer latency indicates a stronger memory of the electric shock in the animal. Compared to the control group, the model group showed a significantly shorter latency (p<0.001), indicating that scopolamine successfully created a memory acquisition impairment model. Compared to the model group, the latency in Example 2 and Example 3 groups was significantly increased (p<0.001, p<0.001), and Example 3 showed a significant increase compared to all control mice (p<0.05).

[0093] The number of errors within 5 minutes is another important indicator for evaluating an animal's memory ability in the platform jumping test. Fewer errors indicate stronger memory. Compared to the control group, the model group had significantly more errors (p<0.001), further demonstrating the success of scopolamine in creating a memory acquisition impairment model. Compared to the model group, the number of errors in each example group was significantly reduced (p<0.05, p<0.001, p<0.001), with Example 3 showing a significant reduction compared to all comparative mice (p<0.05, p<0.05, p<0.01), as shown in Table 6. Figure 4 .

[0094] Table 6. Effects of TSBG on memory acquisition impairment in mice (n=6)

[0095] Note: Compared with the blank group, ### p<0.001; compared with the model group, p<0.01, p<0.001, compared with Example 3, △ p<0.05, △△ p<0.01.

[0096] 2.2.2 Experimental Results of New Object Recognition The Discrimination Index (DI) is an important indicator for evaluating animal memory in novel object recognition experiments; a higher DI value indicates a stronger memory. (See Table 7 and...) Figure 5 As shown, the DI value of the model group was significantly lower than that of the control group (p<0.001), indicating that the model was successfully established. Compared with the model group, the DI of each example group was significantly increased (p<0.01, p<0.001, p<0.001), and the DI of Example 3 was significantly higher than that of all comparative mice (p<0.01, p<0.05, p<0.05).

[0097] Table 7. DI (n=6) of mice in the novel object recognition experiment.

[0098] Note: Compared with the blank group, ### p<0.001; compared with the model group, p<0.01, p<0.001, compared with Example 3, △ p<0.05, △△ p<0.01.

[0099] 2.2.3 Results of the Morris water maze experiment In the water maze experiment, the length of the latency period is an important indicator for evaluating an animal's spatial learning and memory ability; the longer the latency period, the worse the animal's spatial memory ability. (See Table 8 and...) Figure 6 As shown, the water maze results indicated that, compared with the control group, the latency of mice in the model group was significantly increased (p<0.001), indicating that scopolamine can cause spatial memory impairment in mice. Compared with the model group, the latency of groups 2 and 3 was significantly prolonged (p<0.05, p<0.001), and the latency of the positive control group was also significantly prolonged (p<0.001). The latency of group 3 was significantly reduced compared with all control group mice (p<0.01, p<0.05, p<0.05).

[0100] The number of times an animal traverses the platform reflects whether it remembers the platform's location and is an important indicator for assessing spatial memory. Compared with the control group, the number of times the model group mice traversed the platform was significantly reduced (p<0.001), proving the successful establishment of the model. Compared with the model group, the number of times each example group traversed the platform was significantly increased (p<0.01, p<0.001, p<0.001), and Example 3 showed a significant increase compared to all comparative mice (p<0.01, p<0.05, p<0.05).

[0101] Table 8. Effects of TSBG on spatial memory in mice (n=6)

[0102] Note: Compared to the blank group, ### p < 0.001; compared to the model group, p<0.05, p<0.01, p<0.001, compared with Example 3, △ p<0.05, △△ p<0.01.

[0103] In summary, this invention used intraperitoneal injection of scopolamine to establish a mouse model of artificial memory impairment and evaluated the effects of a rare ginsenoside composition on mouse memory impairment. Results from three behavioral tests—the platform jumping test, the novel object recognition test, and the Morris water maze—showed that the rare ginsenoside composition significantly reduced the number of errors and prolonged the latency in the platform jumping test; significantly increased the DI value in the novel object recognition test, effectively enhancing cognitive memory; and significantly increased the number of platform crossings and reduced the latency in the Morris water maze test, effectively improving spatial memory. Therefore, the rare ginsenoside composition has a significant ameliorative effect on mouse memory impairment.

[0104] The above detailed description is a specific illustration of one feasible embodiment of the present invention, and this embodiment is not intended to limit the patent scope of the present invention. It should be noted that all equivalent implementations or modifications made without departing from the present invention should be included within the scope of the technical solution of the present invention. Therefore, the protection scope of the present invention should be determined by the appended claims.

Claims

1. A method of preparing a rare ginsenoside composition, characterized by: Includes the following steps: (1) Take dried ginseng stems and leaves, wash them clean, dry them, crush them, and obtain granules; (2) Take the particles from step (1) and perform enzymatic hydrolysis, ultrasonic extraction, and freeze-drying to obtain ginseng extract; (3) The ginseng extract obtained in step (2) was fermented with yeast and lactobacillus respectively to obtain ginseng fermentation products; (4) The ginseng fermentation product obtained in step (3) was separated and purified by C30 semi-preparative high performance liquid chromatography to obtain the rare ginsenoside composition. The specific operation of step (2) is as follows: Take the particles from step (1) and add 8-15 times the mass of distilled water to obtain a mixed solution; add 0.3%-0.5% of cellulase to the mixed solution, adjust the pH value to 3.5-4.5, and extract under ultrasonic and water bath heating at 60-80℃ for 2-3 h under the condition of vibration power of 200-400 W. Centrifuge and filter to obtain filtrate. Repeat the extraction step 2-3 times, combine the filtrates, freeze dry the filtrate to obtain ginseng extract; The yeast mentioned in step (3) is wine yeast, preservation number: CGMCC 2.3997; the lactobacillus is Lactobacillus acidophilus, preservation number: CGMCC 1.1878; the mass-volume ratio of the ginseng extract and the wine yeast fermentation liquid is 1:1; the mass-volume ratio of the ginseng extract and the Lactobacillus acidophilus fermentation liquid is 1:

1.

2. The method of claim 1, wherein: The specific operation of step (3) is as follows: (3.1) Add distilled water to the ginseng extract obtained in step (2), then add glucose, maltose, casein and ammonium sulfate, sterilize at 115 ℃ for 10-25 min to obtain raw material mixture 1; add wine yeast fermentation liquid to raw material mixture 1 and culture at 28-32 ℃ with shaking for 36-60 h, with a shaking speed of 160-250 rpm / min to obtain ginseng yeast fermentation liquid; (3.2) Add glucose, maltose, casein and ammonium sulfate to the ginseng yeast fermentation broth, adjust the pH to 5.5-7.5, sterilize at 115 ℃ for 10-25 min to obtain raw material mixture 2; add the Lactobacillus acidophilus fermentation broth to the raw material mixture 2 and culture anaerobic or aerobic at 30-37 ℃ for 36-60 h to obtain ginseng Lactobacillus fermentation broth; (3.3) The ginseng lactobacillus fermentation broth obtained in step (3.2) is filtered through diatomaceous earth, the supernatant is collected, and sterilized at 115 °C for 10-25 min to obtain the ginseng fermentation product.

3. The method of claim 2, wherein: In step (3.1), the mass ratio of the ginseng extract to the mixture of glucose, maltose, casein, and ammonium sulfate is 1:0.5-1; the concentrations of glucose, maltose, casein, and ammonium sulfate are 0.01-0.02 g / mL, 0.01-0.02 g / mL, 0.01-0.02 g / mL, and 0.01-0.02 g / mL, respectively. In step (3.2), the mass ratio of the ginseng extract to the mixture of glucose, maltose, casein, and ammonium sulfate is 1:0.5-1; the concentrations of glucose, maltose, casein, and ammonium sulfate are 0.01-0.02 g / mL, 0.01-0.02 g / mL, 0.01-0.02 g / mL, and 0.01-0.02 g / mL, respectively.

4. The method of claim 1, wherein: The specific steps for separation and purification in step (4) are as follows: Chromatographic column: C30 column, 250 mm × 21.2 mm, 5 μm; Column temperature: 28 ℃; Mobile phase A is water, and mobile phase B is 85% acetonitrile; The isocratic elution conditions were 55% B - 45% A; Flow rate: 5 mL / min for 0-30 min, and 2.5 mL / min after 30 min; The detection wavelength is 203 nm; Ginsenoside Rk3 was collected at 34-38 min, ginsenoside Rg5 at 54-59 min, and ginsenoside Rk2 at 85-90 min.

5. The rare ginsenoside composition prepared by the preparation method according to any one of claims 1 to 4, characterized in that: The rare ginsenoside composition is composed of ginsenoside Rk3, ginsenoside Rg5 and ginsenoside Rk2 in a mass ratio of 1-3:4-6:1-3.

6. Use of the rare ginsenoside composition prepared by the preparation method according to any one of claims 1-4 in the preparation of health products that help improve sleep.

7. A health product that helps improve sleep, characterized in that: The health products mentioned above include rare ginsenoside compositions prepared by the preparation method according to any one of claims 1-4.

8. Use of the rare ginsenoside composition prepared by the preparation method according to any one of claims 1-4 in the preparation of a medicine for treating memory disorders.

9. A medicine for treating memory impairment, characterized in that: The medicine comprises a rare ginsenoside composition prepared by the preparation method according to any one of claims 1-4 and pharmaceutically acceptable excipients.

Images