Preparation method and application of ginseng extract containing protopanaxatriol

Abstract

The present invention belongs to the field of plant extraction technology and specifically discloses a method for preparing a ginseng extract containing protopanaxatriol and its application. This invention utilizes a novel processing technique to increase the content of rare saponins while significantly improving the flavor of conventional ginseng extracts. Animal experimental results demonstrate that the ginseng extract of the present invention significantly enhances the sleep-improving efficacy of ginseng. Furthermore, the method of the present invention offers advantages such as high product purity, simple process, low cost, and environmental friendliness, and has broad application prospects in the field of ginseng extraction.

Description

Technical Field

[0001] The invention belongs to the technical field of plant extraction and relates to a preparation method of a ginseng extract containing protopanaxatriol and application thereof. Background Art

[0002] Ginseng is a plant of the genus Panax in the Araliaceae family ( Panax ginseng CA Mayer), has the effects of tonifying the spleen and lungs, dispelling pathogenic factors, replenishing vital energy, calming the mind, relieving palpitations, and improving eyesight and intelligence. Ginsenosides are the main active ingredients of ginseng and can be divided into common ginsenosides and rare ginsenosides. Common ginsenosides generally refer to those naturally occurring in relatively high concentrations in Panax genus plants, primarily including ginsenosides Rb1, Rb2, Rb3, Rc, Rd, Rg1, Rg2, and Re. Rare ginsenosides generally refer to those found in very low concentrations in Panax ginseng plants or detected only in ginseng processed products and metabolites. These include ginsenosides 20(S)-Rh1, 20(R)-Rh1, Rg6, F2, F4, Rk3, Rh4, 20(S)-Rg3, 20(R)-Rg3, Rk1, Rg5, 20(S)-Rh2, 20(R)-Rh2, Compound K(CK), Rk2, Rh3, PPD, and PPT. Studies have shown that rare ginsenosides often exhibit superior pharmacological activity and bioavailability compared to conventional ginsenosides.

[0003] Ginsenoside preparation methods include heating and reflux, water extraction and alcohol precipitation, microwave extraction, and ultrasonic extraction. However, these methods often suffer from high impurity content, complex processes, and low extraction yields of rare saponins. Therefore, there is an urgent need for an extract preparation method that can increase the content of rare saponins and efficiently enrich rare saponin fractions. Summary of the Invention

[0004] To solve at least some of the technical problems in the above-mentioned prior art, the present invention provides a method for preparing a ginseng extract containing protopanaxatriol and its application. Specifically, the present invention includes the following contents.

[0005] A first aspect of the present invention provides a method for preparing a ginseng extract containing protopanaxatriol, comprising the following steps:

[0006] (S1) preparing ginseng, crushing and sieving to obtain ginseng powder, mixing the ginseng powder with a honey solution, and extracting at 50-90° C. for 12-48 hours to obtain an extract (1) and ginseng residue (1);

[0007] (S2) mixing the ginseng residue (1) and the β-galactosidase solution, and extracting at 37-50° C. for 1-10 h to obtain an extract (2) and ginseng residue (2);

[0008] (S3) The ginseng residue (2) is mixed with water and subjected to water extraction at 80-100° C. for 0.5-5 h to obtain an extract (3) and ginseng residue (3), and the extracts (1)-(3) are combined.

[0009] In certain embodiments, according to the method for preparing a ginseng extract containing protopanaxatriol of the present invention, in step (S1), the material-liquid ratio (g / mL) of the crushed ginseng and the honey solution is 1:(1-20).

[0010] In certain embodiments, according to the method for preparing a ginseng extract containing protopanaxatriol of the present invention, the solid-liquid ratio (g / mL) of the ginseng residue (1) to the β-galactosidase solution is 1:(10-40).

[0011] In certain embodiments, according to the method for preparing a ginseng extract containing protopanaxatriol of the present invention, the β-galactosidase solution comprises β-galactosidase.

[0012] In certain embodiments, according to the method for preparing the ginseng extract of protopanaxatriol of the present invention, the solid-liquid ratio (g / mL) of the ginseng residue (2) and water is 1:(5-30).

[0013] In certain embodiments, the method for preparing a ginseng extract containing protopanaxatriol according to the present invention further comprises repeating the water extraction in step (S3).

[0014] The second aspect of the present invention provides a ginseng extract obtained by the preparation method described in the first aspect of the present invention.

[0015] The third aspect of the present invention provides a pharmaceutical composition comprising the ginseng extract according to the second aspect of the present invention and a pharmaceutically acceptable carrier.

[0016] The fourth aspect of the present invention provides use of the ginseng extract according to the second aspect of the present invention in preparing a product for improving sleep.

[0017] In certain embodiments, according to the use of the present invention, the product includes a medicine or a health product.

[0018] The present invention utilizes a novel processing technique to increase the content of rare saponins, particularly 20(S)-PPT, and improve the flavor of conventional ginseng extracts. The ginseng extracts of the present invention enhance the sleep-improving effects of ginseng. Furthermore, the method of the present invention offers advantages such as high product purity, simple process, low cost, and environmental friendliness, and has broad application prospects in the field of ginseng extraction. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 A standard curve of total saponins is shown.

[0020] Figure 2 The liquid phase diagram of the standard is shown.

[0021] Figure 3 It shows that sample No. Ⅰ does not contain the rare saponin 20(S)-PPT.

[0022] Figure 4 It shows that sample II contains the rare saponin 20(S)-PPT.

[0023] Figure 5 This indicates that the ginseng extract obtained by enzymatic extraction alone does not contain the rare saponin 20(S)-PPT.

[0024] Figure 6 This indicates that the ginseng extract obtained by extracting honey alone does not contain the rare saponin 20(S)-PPT.

[0025] Figure 7 The differences in odor of different ginseng extracts are shown.

[0026] Figure 8 Shown are the sleep time profiles of female fruit flies in different treatment groups over a 24-hour period, with sleep recorded every 30 minutes during the day (LP) and night (DP).

[0027] Figure 9 Shown are the total sleep time of female flies in different treatment groups during a 24-hour period.

[0028] Figure 10 Shown are the sleep times of female fruit flies in different treatment groups during a 24-hour period.

[0029] Figure 11 Shown are the average sleep bout durations during a 24-hour period for female flies in the different treatment groups. DETAILED DESCRIPTION

[0030] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0031] It should be understood that the terms described in the present invention are only for describing particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges in the present invention, it should be understood that the upper and lower limits of the ranges and each intermediate value therebetween are specifically disclosed. Each smaller range between any stated value or intermediate value within a stated range and any other stated value or intermediate value within the stated range is also included in the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0032] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the invention belongs. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In the event of any conflict with any incorporated document, the content of this specification shall prevail.

[0033] Preparation method

[0034] One aspect of the present invention provides a method for preparing a ginseng extract containing protopanaxatriol, wherein the protopanaxatriol is a rare saponin 20(S)-PPT.

[0035] The present invention has found that when only honey is used for extraction or when enzymatic hydrolysis is used for extraction, the content of rare saponins is low and 20(S)-PPT rare saponin is not extracted. However, combining the two steps can synergistically increase the content of rare saponins and extract 20(S)-PPT rare saponin. The 20(S)-PPT rare saponin has the structure shown in Formula I:

[0036] Formula I.

[0037] In a preferred embodiment, the preparation method of the present invention comprises the following steps:

[0038] (S1) preparing ginseng, crushing and sieving to obtain ginseng powder, mixing the ginseng powder with a honey solution, and extracting at 50-90° C., for example, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90° C., for 12-48 h, for example, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48 h, to obtain an extract (1) and ginseng residue (1);

[0039] (S2) mixing the ginseng residue (1) and the β-galactosidase solution, and extracting at 37-50° C., for example, 37, 38, 39, 40, 42, 44, 46, 48, 50° C., for 1-10 h, for example, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10 h, to obtain an extract (2) and ginseng residue (2);

[0040] (S3) The ginseng residue (2) is mixed with water and extracted at 80-100°C, preferably 82-100°C, also preferably 84-100°C, further preferably 86-100°C, more preferably 88-100°C, for example, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100°C for 0.5-5 h, preferably 0.5-4.5 h, also preferably 0.5-4 h, further preferably 0.5-3.5 h, more preferably 0.5-3 h, for example, 0.5, 1, 1.5, 2, 2.5, 3 h to obtain an extract (3) and ginseng residue (3), and the extracts (1)-(3) are combined.

[0041] It is understood that in order to improve the extraction efficiency, stirring, ultrasonic treatment and the like can be used during the extraction process, and the extraction steps can be repeated.

[0042] In order to increase the content of rare saponins in the ginseng extract, the material-liquid ratio of the ginseng crushed material and the honey solution can be controlled within an appropriate range. In a preferred embodiment, the material-liquid ratio of the ginseng crushed material and the honey is 1:(1-20), preferably 1:(1-18), further preferably 1:(1-16), further preferably 1:(1-14), more preferably 1:(1-12), more preferably 1:(1-10), for example 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10.

[0043] In the present invention, the concentration of the honey solution is preferably 30-100%, more preferably 35-100%, further preferably 40-100%, more preferably 45-100%, and more preferably 50-100%, for example 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100%. The honey solution of the present invention is not particularly limited, and examples thereof include but are not limited to 100% pure honey, a solution of honey in fructose syrup (the ratio of honey to fructose syrup is not particularly limited), and the like. The honey of the present invention is not particularly limited, and examples thereof include but are not limited to linden honey, sophora flower honey, jujube flower honey, lychee honey, vitex honey, buckwheat honey, and the like. In a preferred embodiment, the honey is 100% pure linden honey. In another preferred embodiment, the honey is 100% pure sophora flower honey. In yet another preferred embodiment, the honey solution is a solution obtained by mixing honey and fructose syrup in a 1:1 ratio.

[0044] In order to increase the content of rare saponins in the ginseng extract, the material-liquid ratio of the ginseng residue (1) to the β-galactosidase solution can be controlled within an appropriate range. In a preferred embodiment, the material-liquid ratio of the ginseng residue (1) to the β-galactosidase solution is 1:(10-40), preferably 1:(10-35), and further preferably 1:(10-30), for example, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:21, 1:22, 1:23, 1:24, 1:25, 1:26, 1:27, 1:28, 1:29, 1:30.

[0045] In the present invention, the β-galactosidase solution includes β-galactosidase (sometimes also called β-galactosidase mixed enzyme due to different sources). The enzymatic activity of the β-galactosidase is not less than 10,000 U / g, and its source is not particularly limited. It can be purchased from commercially available products or produced by suitable strains. Examples of the strains include but are not limited to Bacillus subtilis, Kluyveromyces lactis, etc. The solvent of the β-galactosidase solution is not particularly limited, as long as the activity of the β-galactosidase can be maintained. Examples of the solvent include but are not limited to sodium citrate, citric acid, phosphate buffer, Tris-HCl buffer, normal saline, etc. In a preferred embodiment, the β-galactosidase solution includes 50-400 mg of β-galactosidase, 0.01-1 M sodium citrate and / or citric acid, for example, 10-100 mL of 0.1 M sodium citrate and / or 20-120 mL of 0.1 M citric acid.

[0046] In order to increase the content of rare saponins in ginseng extract, the material-liquid ratio of the ginseng residue (2) and water can be controlled to 1:(5-30), preferably 1:(5-25), further preferably 1:(5-20), and more preferably 1:(5-15), such as 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, and 1:15.

[0047] Ginseng extract

[0048] One aspect of the present invention provides a ginseng extract obtained according to the preparation method of the present invention.

[0049] In a preferred embodiment, the ginseng extract of the present invention contains 20(S)-Rg3 rare saponins in an amount of not less than 1653.837099 μg / g of crude drug, F2 rare saponins in an amount of not less than 1229.27621 μg / g of crude drug, 20(S)-PPT rare saponins in an amount of not less than 8.016517 μg / g of crude drug, Rk1 rare saponins in an amount of not less than 325.226528 μg / g of crude drug, Rg5 rare saponins in an amount of not less than 601.797812 μg / g of crude drug, 20(S)-Rh2 rare saponins in an amount of not less than 135.552609 μg / g of crude drug, and 20(R)-Rh2 rare saponins in an amount of not less than 202.171221 μg / g of crude drug.

[0050] Pharmaceutical composition

[0051] In one aspect, the present invention provides a pharmaceutical composition comprising the ginseng extract according to the present invention and a pharmaceutically acceptable carrier. The pharmaceutical composition of the present invention can be used to improve sleep, including but not limited to the following: increasing total sleep time, reducing sleep frequency, shortening sleep onset time, improving sleep continuity, increasing deep sleep time, stabilizing sleep rhythms, and improving sleep quality.

[0052] In the present invention, a pharmaceutically acceptable carrier is involved in carrying or delivering a drug from one organ or part of the body to another. Each carrier is "acceptable" if it is compatible with the other ingredients of the formulation (e.g., ginseng extract) and does not harm the patient. Pharmaceutically acceptable carriers include at least one of a diluent, a filler, an absorbent, a wetting agent, a binder, a disintegrant, a lubricant, a sweetener, a preservative, and an antioxidant. Examples of diluents include, but are not limited to, physiological saline, aqueous buffer solutions, solvents, dispersion vehicles, etc.; fillers include, but are not limited to, starch, lactose, mannitol, microcrystalline cellulose, etc.; absorbents include, but are not limited to, calcium sulfate, calcium hydrogen phosphate, calcium carbonate, etc.; wetting agents include, but are not limited to, water, ethanol, etc.; binders include, but are not limited to, hydroxypropyl methylcellulose, povidone, microcrystalline cellulose, etc.; disintegrants include, but are not limited to, cross-linked sodium carboxymethyl cellulose, cross-linked povidone, surfactants, low-substituted hydroxypropyl cellulose, etc.; lubricants include, but are not limited to, magnesium stearate, talc, polyethylene glycol, sodium lauryl sulfate, micropowdered silica gel, talc, etc.; sweeteners include, but are not limited to, sucralose, acesulfame, saccharin, sucrose, xylitol, mannitol, sorbitol, glucose, fructose, aspartame, etc.; preservatives include, but are not limited to, parabens, chlorobutanol, phenol, sorbic acid, etc.; antioxidants include, but are not limited to, ascorbic acid, methionine, etc.

[0053] application

[0054] One aspect of the present invention provides the use of the ginseng extract according to the present invention in the preparation of a product for improving sleep. Examples of the product include, but are not limited to, medicines, health supplements, and the like.

[0055] Example

[0056] The following describes a method for increasing the content of rare saponins in ginseng extract and its application.

[0057] 1. Preparation of Ginseng Extract

[0058] 1. Conventional Extraction Method

[0059] Ginseng was crushed, sieved, and extracted with water twice. The first time, the solid-liquid ratio was 1:10, and the extraction was carried out for 1 hour. The second time, the solid-liquid ratio was 1:8, and the extraction was carried out for 1 hour. The two extracts were combined, concentrated, and set aside. It was named Sample No. Ⅰ.

[0060] 2. Extraction methods to improve flavor and increase rare saponin content

[0061] Ginseng was crushed and sieved, and linden honey (alpine linden honey produced by Jilin Aodong Health Technology Co., Ltd., production date: 20240129) was added at a material-liquid ratio of 1:5. The mixture was kept at 85℃ and ultrasonicated for 36 hours. The ginseng was filtered out and the filtrate ① was set aside. The ginseng residue was enzymatically hydrolyzed with food-grade lactase (β-galactosidase, derived from Bacillus subtilis) (0.1 M food-grade sodium citrate 40 mL, 0.1 M food-grade citric acid 60 mL, 200 mg lactase) at a material-liquid ratio of 1:20. The optimal temperature for lactase was 50℃ for enzymatic hydrolysis for three hours. After the enzymatic hydrolysis was completed, the filtrate ② was set aside. The ginseng residue was extracted twice with deionized water at a material-liquid ratio of 1:10, each time for 1 hour, filtered, and the filtrate ③ was combined for later use. The filtrates ①, ②, and ③ were combined, the enzyme was inactivated at high temperature, concentrated, and set aside. It was named sample No. II.

[0062] 3. Single honey extraction method

[0063] Ginseng was crushed and sieved, and linden honey (alpine linden honey produced by Jilin Aodong Health Technology Co., Ltd., production date: 20240129) was added at a solid-liquid ratio of 1:5. The mixture was kept warm at 85°C and ultrasonicated for 36 hours. The ginseng residue was filtered out and the filtrate was set aside. The ginseng residue was extracted twice with deionized water at a solid-liquid ratio of 1:10, each time for 1 hour. The extracts were filtered and the filtrates were combined for later use and named as sample III.

[0064] 4. Single Enzymatic Extraction Method

[0065] Ginseng was crushed and sieved, and enzymatically hydrolyzed using food-grade lactase (β-galactosidase, derived from Bacillus subtilis) with a solid-liquid ratio of 1:20 (40 mL of 0.1 M food-grade sodium citrate, 60 mL of 0.1 M food-grade citric acid, and 200 mg of lactase). The optimal temperature for lactase was 50°C for three hours. After the enzymatic hydrolysis was completed, the filtrate was filtered and the filtrate was set aside. The ginseng residue was extracted twice with deionized water at a solid-liquid ratio of 1:10, each time for 1 hour. The filtrate was combined and set aside, and named sample IV.

[0066] 2. Detection of total saponin content in different ginseng extracts

[0067] 1. Preparation of Reference Solution

[0068] Take an appropriate amount of ginsenoside Re reference substance, accurately weigh it, and add methanol to make a solution containing 1 mg per 1 mL.

[0069] 2. Test Sample Preparation

[0070] Samples No. Ⅰ and No. Ⅱ were concentrated to a small volume and extracted twice with water-saturated n-butanol at a volume ratio of 1:3. The solvent was recovered, dried, and dissolved by ultrasonication in methanol.

[0071] 3. Preparation of the Standard Curve

[0072] Accurately pipette 20 μL, 40 μL, 80 μL, 120 μL, 160 μL, and 200 μL of the reference solution into stoppered test tubes, evaporate the solvent at low temperature, add 0.5 mL of 1% vanillin perchloric acid test solution, mix thoroughly in a 60°C water bath, and heat for 15 minutes. Immediately cool with ice water for 2 minutes, add 5 mL of 77% sulfuric acid solution, and shake well. Use the reagent as a blank. After eliminating bubbles, measure the absorbance at a wavelength of 540 nm using UV-visible spectrophotometry. Plot a standard curve with absorbance as the y-axis and concentration as the abscissa.

[0073] 4. Test Results

[0074] The total saponin content of sample No. I and sample No. II was tested. The results are shown in Table 1. The total saponin content of sample No. II was 1.45 times that of sample No. I.

[0075] Table 1 Total saponin content (n=3)

[0076]

[0077] 3. Detection of Monomeric Saponin Content in Different Ginseng Extracts

[0078] 1. Preparation of Mixed Reference Solution

[0079] Accurately weigh appropriate amounts of 20(S)-Rg3 rare saponin, F2 rare saponin, 20(S)-PPT rare saponin, Rk1 rare saponin, Rg5 rare saponin, 20(S)-Rh2 rare saponin, and 20(R)-Rh2 rare saponin, dissolve them in methanol to prepare single reference solutions. Take appropriate amounts of the single reference solutions to prepare mixed reference solutions containing the above components, with concentrations of 0.389, 0.389, 0.061, 0.033, 0.030, 0.05, and 0.033 mg / mL, respectively.

[0080] 2. Chromatographic Conditions

[0081] Chromatographic column: Agilent ZORBAX SB-Aq C18 column (250 mm × 4.6 mm, 5 μm); mobile phase: 0.1% phosphoric acid aqueous solution (A)-acetonitrile (B), elution gradient see Table 2 below; flow rate: 1 mL / min; column temperature: 35°C; detection wavelength: 203 nm; injection volume: 10 μL.

[0082] Table 2 Elution procedure

[0083]

[0084] 3. Test results

[0085] The monomer saponin content of sample No. I, sample No. II, sample No. III and sample No. IV was tested, and the results are shown in Tables 3 and 4. The rare saponin content of sample No. II was 9.38 times that of sample No. I, 5.93 times that of sample No. III, and 2.38 times that of sample No. IV.

[0086] Table 3 Contents of some rare saponins in samples I and II

[0087]

[0088] Table 4 Contents of some rare saponins in samples III and IV

[0089]

[0090] 4. Odor Differences of Different Ginseng Extracts

[0091] An electronic nose technique was used to detect flavor differences. The surfaces of 18 metal oxide semiconductor (MOS) sensors were confirmed to be free of contamination. A dedicated cleaning cotton swab was used to gently wipe the sensor chamber. After turning on the device, a 20-minute preheating period was performed to ensure sensor stability (baseline drift should be <±2%). The gas sampling system was configured by connecting a nitrogen carrier gas tank and adjusting the flow rate to 1 L / min. The sample collection time was 60 seconds, followed by a 2-minute rinse. Samples I and II were configured with the same crude drug concentration of 1 g / 10 mL. 3 mL of sample was added to each sample bottle and allowed to stand for 4 hours. The sample was then tested on the instrument, the maximum odor value was selected, the data was loaded, and the top eight sensors with the greatest sensor differences were selected. A radar chart was created and PCA analysis was performed.

[0092] The analysis of electronic nose results combined with PCA results showed that there were significant differences in the odor of samples No. Ⅰ, No. Ⅱ and No. Ⅲ, especially in terms of alcohol and ketone aldehyde components such as those representing sweetness, fruitiness and fragrance. Sample No. Ⅱ was significantly stronger than samples No. Ⅰ and No. Ⅲ.

[0093] 5. Taste Differences of Different Ginseng Extracts

[0094] The sensory evaluation method was conducted in accordance with the national standard "Botanical Beverages" (GB / T31326-2014). Samples I and II were prepared with 50 mL of ginseng raw material containing 1 g, sterilized, and immersed in a transparent glass for 5 minutes while stirring. A panel of 10 trained judges (4 men and 6 women, aged 23-45 years) conducted a sensory evaluation of the two ginseng beverages. The judges scored the appearance, color, taste, ginseng flavor, and odor on a scale of 0-100, with a total score of 4000, representing the lowest to highest score.

[0095] Table 5 Sensory evaluation of different ginseng extracts

[0096]

[0097] The total score of sample No. Ⅰ was 2276 points, and that of sample No. Ⅱ was 3204 points. The flavor of ginseng extract was effectively improved after processing.

[0098] 6. Effects of different ginseng extracts on improving sleep

[0099] 1. Drosophila sleep monitoring system

[0100] This example uses the DAMS fruit fly activity monitoring system. Each fruit fly is placed in a separate monitoring tube, which is then inserted into the center of the DAM2 fruit fly activity monitor. The monitor emits an infrared beam during monitoring. As a fruit fly moves within the tube, it interrupts the infrared beam. Each interruption is detected and automatically counted by the monitor. A single monitor can simultaneously monitor the sleep activity of 32 fruit flies. During the experiment, the monitor was placed in a constant temperature and humidity incubator set to a 12h:12h light:dark cycle, with an incubation temperature of (25±1)°C and a relative humidity of 60%. The sleep activity of the fruit flies was recorded.

[0101] 2. Criteria for determining Drosophila sleep

[0102] The DAMS data acquisition software automatically records the number of times a fruit fly blocks the infrared light beam every 5 minutes. If the number of fruit fly activities within these 5 minutes is zero, the fruit fly is determined to be in a resting state, which meets the criteria for sleep behavior. The fruit fly is determined to be in a sleeping state and its sleep time is included in the fruit fly's sleep time. Based on this, the average sleep time of the fruit fly can be further calculated.

[0103] 3. Drosophila Sleep Deprivation Method

[0104] The DAM2 Drosophila Activity Monitor is mounted on an oscillator and the oscillation time and interval are set. During the oscillation, the fruit flies in the monitoring tube vibrate along with the detector and cannot remain still, thus achieving mechanical sleep deprivation.

[0105] Experimental groups: control group (Control), 32 rats with an equal amount of RO water added to the basal culture medium; model group / sleep deprivation group (SD), 32 rats with an equal amount of RO water added to the basal culture medium; sample No. I (I), 32 rats with a drug-containing culture medium prepared according to a crude drug dosage of 1 mg / mL; sample No. II (II), 32 rats with a drug-containing culture medium prepared according to a crude drug dosage of 1 mg / mL.

[0106] 4. Drosophila sleep monitoring method

[0107] Female fruit flies were randomly divided into four groups and placed in monitoring tubes containing culture medium, which were centrally inserted into a DAM2 Drosophila activity monitor. Each group had one monitor (n=32). Except for the control group, the monitors in the other three groups were placed on an oscillator that oscillated for 5 seconds per minute. The control, SD, and SD+drug-treated groups were all placed in a 12-h:12-h light:dark cycle, and sleep patterns were recorded for 72 hours.

[0108] 5. Statistical Analysis

[0109] Data were analyzed using software, and one-way ANOVA and t-test (comparison between two groups) were used to analyze the data and expressed as Mean ± SD. P < 0.05 was considered to indicate a statistically significant difference. All experiments were repeated at least three times.

[0110] 6. Experimental Results

[0111] 6.1 Circadian Rhythm in Female Drosophila

[0112] The fruit fly's circadian activity responds to light switching, with peaks in activity both after the lights are on and off, indicating a normal wild-type circadian rhythm. Compared to the model group, female fruit flies fed samples I and II showed a weakened circadian rhythm, with the weakening being more pronounced in the sample II group.

[0113] 6.2 Sleep duration of female fruit flies

[0114] The results are as follows Figure 9 As shown in the data, the sleep time of female fruit flies was significantly reduced after sleep deprivation, and the total sleep time of female fruit flies fed with sample No. Ⅰ and sample No. Ⅱ was significantly increased, and the effect of feeding sample No. Ⅱ was more significant (compared with the control group, #P<0.05; compared with the model group, *P<0.05, ***P<0.001; compared with sample No. Ⅰ and sample No. Ⅱ, &P<0.05).

[0115] 6.3 Sleep frequency of female fruit flies

[0116] The results are as follows Figure 10 As shown in the results, sleep deprivation significantly increased the number of sleep episodes in female fruit flies. When fed Samples I and II, the total number of sleep episodes in female fruit flies was significantly reduced. This result indicates that both samples can effectively improve sleep fragmentation in fruit flies. The improvement was even more significant in fruit flies fed Sample II (compared with the control group, #P<0.05; compared with the model group, *P<0.05, ***P<0.001; compared with Samples I and II, &P<0.05).

[0117] 6.4 Average sleep bout duration in female Drosophila

[0118] The results are as follows Figure 11 As shown, sleep deprivation significantly shortened the average duration of sleep bouts in female fruit flies. The average duration of sleep bouts in female fruit flies fed with sample No. Ⅰ and sample No. Ⅱ increased significantly, indicating that both samples can effectively improve the sleep quality of fruit flies, and the improvement in sleep duration in fruit flies fed with sample No. Ⅱ was more significant (compared with the control group, #P<0.05; compared with the model group, **P<0.01, ***P<0.001; compared with sample No. Ⅰ and sample No. Ⅱ, &P<0.05).

[0119] Finally, it should be noted that the above embodiments are intended only to illustrate the technical solutions of the present invention, and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that the technical solutions described in the above embodiments may be modified or some of the technical features thereof may be replaced with equivalents. Such modifications or replacements do not deviate from the spirit and scope of the technical solutions of the various embodiments of the present invention.

Claims

1. A method for preparing a ginseng extract containing protopanaxatriol, characterized in that: The protopanaxantriol is 20(S)-PPT, and the preparation method comprises the following steps: (S1) preparing ginseng, crushing and sieving to obtain a ginseng crushed material, mixing the ginseng crushed material with a honey solution, and extracting at 50-85° C. for 12-48 hours to obtain a first extract and a first ginseng residue, wherein the material-liquid ratio of the ginseng crushed material to the honey solution is 1:(5-20); (S2) mixing the first ginseng residue and the β-galactosidase solution, and extracting at 50° C. for 3-10 hours to obtain a second extract and a second ginseng residue, wherein the material-liquid ratio of the first ginseng residue to the β-galactosidase solution is 1:(10-30); (S3) mixing the second ginseng residue with water, extracting the mixture with water at 88-100° C. for 0.5-3 h to obtain a third extract and a third ginseng residue, and combining the first, second and third extracts, wherein the material-to-liquid ratio of the second ginseng residue to water is 1:

10.

2. The method for preparing the ginseng extract containing protopanaxatriol according to claim 1, wherein: In step (S2), the β-galactosidase solution includes β-galactosidase.

3. The method for preparing the ginseng extract containing protopanaxatriol according to claim 1, wherein: The method further comprises repeating the water extraction in step (S3).

4. Use of the preparation method of the ginseng extract containing protopanaxatriol according to any one of claims 1 to 3 in the preparation of a drug for improving sleep.

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