Preparation method and application of ginseng extract containing protopanaxatriol

Abstract

The invention belongs to the technical field of plant extraction, and particularly discloses a preparation method and application of a ginseng extract containing protopanaxatriol. By adopting a new processing technology, the content of rare saponin is increased, and meanwhile, the flavor of the conventional ginseng extracting solution is greatly improved. Animal experiment results prove that the ginseng extracting solution remarkably improves the sleep improving effect of ginseng. In addition, the method disclosed by the invention has the advantages of high product purity, simple process, low cost, environmental protection and the like, and has a wide application prospect in the field of ginseng extraction.

Description

Technical Field

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

[0002] Ginseng is a plant of the genus Panax in the family Araliaceae ( Panax ginseng C.A. Mayer), and has the effects of invigorating the spleen and lungs, expelling pathogenic factors, greatly replenishing vital energy, calming the mind, arresting palpitation, and improving eyesight and intelligence. Ginsenosides are important active components of ginseng, and can be divided into common ginsenosides and rare ginsenosides. Common ginsenosides generally refer to ginsenosides that naturally exist and have relatively high contents in plants of the genus Panax, mainly including ginsenoside Rb1, Rb2, Rb3, Rc, Rd, Rg1, Rg2, Re, etc. Rare ginsenosides generally refer to ginsenosides with very low contents in plants of the genus Panax or only detectable in processed products and metabolites of ginseng, mainly including ginsenoside 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, PPT, etc. Research shows that compared with conventional ginsenosides, rare ginsenosides often have better pharmacological activities and bioavailability.

[0003] The preparation methods of ginsenosides include heating reflux method, water extraction and alcohol precipitation method, microwave extraction method, ultrasonic extraction method, etc. However, there are problems such as high impurity content in the product, cumbersome process, and low extraction rate of rare ginsenosides. Therefore, there is an urgent need for an extract preparation method that can increase the content of rare ginsenosides and efficiently enrich the rare ginsenoside part. Summary of the Invention

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

[0005] In a first aspect of the present invention, there is provided a preparation method of a ginseng extract containing protopanaxatriol, which includes the following steps: (S1) Prepare ginseng, crush and sieve it to obtain a ginseng powder, mix the ginseng powder with a honey solution, and extract at 50 - 90 °C for 12 - 48 h to obtain an extract (1) and ginseng residue (1); (S2) Mix the ginseng residue (1) with a β-galactosidase solution, and extract at 37 - 50 °C for 1 - 10 h to obtain an extract (2) and ginseng residue (2); (S3) Mix the ginseng residue (2) with water, perform water extraction at 80 - 100 °C for 0.5 - 5 h to obtain an extract (3) and ginseng residue (3), and combine extracts (1) - (3).

[0006] In certain embodiments, for the method for preparing a ginseng extract containing protopanaxatriol according to the present invention, in step (S1), the material - liquid ratio (g / mL) of the pulverized ginseng and the honey solution is 1:(1 - 20).

[0007] In certain embodiments, for the method for preparing a ginseng extract containing protopanaxatriol according to the present invention, the material - liquid ratio (g / mL) of the ginseng residue (1) and the β - galactosidase solution is 1:(10 - 40).

[0008] In certain embodiments, for the method for preparing a ginseng extract containing protopanaxatriol according to the present invention, the β - galactosidase solution comprises β - galactosidase.

[0009] In certain embodiments, for the method for preparing a ginseng extract of protopanaxatriol according to the present invention, the material - liquid ratio (g / mL) of the ginseng residue (2) and water is 1:(5 - 30).

[0010] In certain embodiments, in the method for preparing a ginseng extract containing protopanaxatriol according to the present invention, it further includes repeating the water extraction in step (S3).

[0011] In a second aspect of the present invention, there is provided a ginseng extract obtained by the preparation method described in the first aspect of the present invention.

[0012] In a third aspect of the present invention, there is provided a pharmaceutical composition comprising the ginseng extract described in the second aspect of the present invention and a pharmaceutically acceptable carrier.

[0013] In a fourth aspect of the present invention, there is provided the use of the ginseng extract described in the second aspect of the present invention in the preparation of a product for improving sleep.

[0014] In certain embodiments, for the use according to the present invention, the product includes drugs and health products.

[0015] The present invention increases the content of rare saponins, especially the content of 20(S) - PPT rare saponins, by adopting a new processing technology and improves the flavor of the conventional ginseng extract. The ginseng extract of the present invention enhances the efficacy of ginseng in improving sleep. In addition, the method of the present invention has the advantages of high product purity, simple process, low cost, environmental protection, etc., and has broad application prospects in the field of ginseng extraction. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 Shows the standard curve of total saponins.

[0017] Figure 2 Shows the HPLC chromatogram of the reference substance.

[0018] Figure 3 Shows that sample No. Ⅰ does not contain 20(S)-PPT rare saponins.

[0019] Figure 4 Shows that sample No. Ⅱ contains 20(S)-PPT rare saponins.

[0020] Figure 5 Shows that the ginseng extract obtained by enzymatic hydrolysis alone does not contain 20(S)-PPT rare saponins.

[0021] Figure 6 Shows that the ginseng extract obtained by honey extraction alone does not contain 20(S)-PPT rare saponins.

[0022] Figure 7 Shows the odor differences of different ginseng extracts.

[0023] Figure 8 Shows the sleep time curves of female Drosophila in different treatment groups during 24 hours, recorded every 30 minutes during the day (LP) and at night (DP).

[0024] Figure 9 Shows the total sleep time of female Drosophila in different treatment groups during 24 hours.

[0025] Figure 10 Shows the number of sleep episodes of female Drosophila in different treatment groups during 24 hours.

[0026] Figure 11 Shows the average sleep episode duration of female Drosophila in different treatment groups during 24 hours. Detailed Description of the Invention

[0027] The various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0028] It should be understood that the terms used in the present invention are only for describing specific embodiments and are not intended to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that the upper and lower limits of the range and each intermediate value therebetween are specifically disclosed. Each intermediate value within any stated value or stated range, as well as each smaller range between 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 from the range.

[0029] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although this invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of this invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the said documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0030] Preparation method In one aspect of this invention, there is provided a method for preparing a ginseng extract containing protopanaxatriol, wherein protopanaxatriol is the rare saponin 20(S)-PPT.

[0031] It has been found through research in this invention that when only honey extraction or enzymatic hydrolysis is carried out alone, the content of rare saponins is relatively low, and 20(S)-PPT rare saponins cannot be extracted. However, combining the two steps can synergistically increase the content of rare saponins and extract 20(S)-PPT rare saponins, wherein 20(S)-PPT rare saponins have the structure shown in Formula I: Formula I.

[0032] In a preferred embodiment, the preparation method of this invention comprises the following steps: (S1) Prepare ginseng, crush it and sieve to obtain ginseng powder, mix the ginseng powder with a honey solution, and extract at 50 - 90 °C, such as 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, such as 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48 h, to obtain extraction solution (1) and ginseng residue (1); (S2) Mix the ginseng residue (1) with a β-galactosidase solution, and extract at 37 - 50 °C, such as 37, 38, 39, 40, 42, 44, 46, 48, 50 °C for 1 - 10 h, such as 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 extraction solution (2) and ginseng residue (2); (S3) Mix the ginseng residue (2) with water and perform water extraction at 80 - 100 °C, preferably 82 - 100 °C, more preferably 84 - 100 °C, further preferably 86 - 100 °C, still 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, more preferably 0.5 - 4 h, further preferably 0.5 - 3.5 h, still 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 combine extracts (1) - (3).

[0033] It can be understood that, in order to improve the extraction efficiency, treatments such as stirring and ultrasonic treatment can be adopted during the extraction process, and moreover, the extraction steps can be repeated.

[0034] In order to increase the content of rare saponins in the ginseng extract, the material - liquid ratio of the ginseng powder and the honey solution can be controlled within a suitable range. In a preferred embodiment, the material - liquid ratio of the ginseng powder and the honey is 1:(1 - 20), preferably 1:(1 - 18), more preferably 1:(1 - 16), further preferably 1:(1 - 14), still more preferably 1:(1 - 12), still 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.

[0035] In the present invention, the concentration of the honey solution is preferably 30 - 100%, more preferably 35 - 100%, further preferably 40 - 100%, still more preferably 45 - 100%, still more preferably 50 - 100%, for example 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100%. The honey solution of the present invention is not particularly limited, and its examples include but are not limited to 100% pure honey, a fructose - glucose syrup solution of honey (the ratio of honey and fructose - glucose syrup is not particularly limited), etc. The honey of the present invention is not particularly limited, and its examples include but are not limited to linden honey, pagoda tree honey, jujube honey, litchi honey, vitex honey, buckwheat honey, etc. In a preferred embodiment, the honey is 100% pure linden honey. In another preferred embodiment, the honey is 100% pure pagoda tree honey. In yet another preferred embodiment, the honey solution is a solution obtained by mixing honey and fructose - glucose syrup in a 1:1 ratio.

[0036] To increase the content of rare saponins in ginseng extract, the material-liquid ratio of the ginseng residue (1) and the β-galactosidase solution can be controlled within a suitable range. In a preferred embodiment, the material-liquid ratio of the ginseng residue (1) and the β-galactosidase solution is 1:(10 - 40), preferably 1:(10 - 35), more preferably 1:(10 - 30), such as 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.

[0037] In the present invention, the β-galactosidase solution includes β-galactosidase (sometimes also called β-galactosidase mixture due to different sources). The enzyme 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 it can maintain the activity of β-galactosidase. Examples of the solvent include but are not limited to sodium citrate, citric acid, phosphate buffer, Tris-HCl buffer, physiological saline, etc. In a preferred embodiment, the β-galactosidase solution includes 50 - 400 mg of β-galactosidase, 0.01 - 1 M of 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.

[0038] 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 be 1:(5 - 30), preferably 1:(5 - 25), more preferably 1:(5 - 20), even 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, 1:15.

[0039] Ginseng extract In one aspect of the present invention, there is provided a ginseng extract obtained according to the preparation method of the present invention.

[0040] In a preferred embodiment, the ginseng extract of the present invention contains not less than 1653.837099 μg / g of crude drug amount of 20(S)-Rg3 rare saponin, not less than 1229.27621 μg / g of crude drug amount of F2 rare saponin, not less than 8.016517 μg / g of crude drug amount of 20(S)-PPT rare saponin, not less than 325.226528 μg / g of crude drug amount of Rk1 rare saponin, not less than 601.797812 μg / g of crude drug amount of Rg5 rare saponin, not less than 135.552609 μg / g of crude drug amount of 20(S)-Rh2 rare saponin, and not less than 202.171221 μg / g of crude drug amount of 20(R)-Rh2 rare saponin.

[0041] Drug composition One aspect of the present invention provides a drug composition, which comprises the ginseng extract according to the present invention and a pharmaceutically acceptable carrier. The drug composition of the present invention can be used to improve sleep, and the improvement of sleep includes, but is not limited to, the following situations: increasing the total sleep time, reducing the number of awakenings, shortening the sleep onset time, improving sleep continuity, increasing the deep sleep time, stabilizing the sleep rhythm, improving the sleep quality, etc.

[0042] In the present invention, the pharmaceutically acceptable carrier is involved in transporting or delivering the drug from one organ or part of the body to another organ or part of the body. Each carrier is "acceptable", that is, it is compatible with other components of the formulation (such as ginseng extract) and does not harm the patient. The pharmaceutically acceptable carrier includes at least one of diluents, fillers, absorbents, wetting agents, binders, disintegrants, lubricants, sweeteners, preservatives, and antioxidants. Among them, examples of diluents include, but are not limited to, physiological saline, aqueous buffer solutions, solvents, dispersion media, 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, hypromellose, povidone, microcrystalline cellulose, etc.; disintegrants include, but are not limited to, croscarmellose sodium, crospovidone, surfactants, low-substituted hydroxypropyl cellulose, etc.; lubricants include, but are not limited to, magnesium stearate, talc, polyethylene glycol, sodium lauryl sulfate, colloidal silicon dioxide, talc, etc.; sweeteners include, but are not limited to, sucralose, acesulfame potassium, 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.

[0043] Application 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 such products include, but are not limited to, drugs, health products, etc.

[0044] Examples The following shows a method for increasing the content of rare saponins in ginseng extract and its application.

[0045] I. Preparation of ginseng extract 1. Conventional extraction method Ginseng is crushed, sieved, and extracted with water twice. For the first extraction, the solid-liquid ratio is 1:10 and the extraction time is 1 hour. For the second extraction, the solid-liquid ratio is 1:8 and the extraction time is 1 hour. The two extraction liquids are combined, concentrated, and reserved for use, named Sample No. I.

[0046] 2. Extraction method for improving flavor and increasing the content of rare saponins Ginseng is crushed, sieved, and added with linden honey (Alpine Linden Honey produced by Jilin Aodong Health Technology Co., Ltd., production date: 20240129) at a solid-liquid ratio of 1:5, and kept at 85°C with ultrasonic treatment for 36 hours. The ginseng is filtered out, and the filtrate ① is reserved for use. The ginseng residue is enzymatically hydrolyzed with an enzymatic hydrolysate of food-grade lactase (β-galactosidase, sourced from Bacillus subtilis) (prepared by mixing 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) at a solid-liquid ratio of 1:20. The optimal temperature of lactase for enzymatic hydrolysis is 50°C for 3 hours. After the enzymatic hydrolysis, filtration is carried out, and the filtrate ② is reserved for use. The ginseng residue is extracted with deionized water twice at a solid-liquid ratio of 1:10, each time for 1 hour, filtered, and the combined filtrate ③ is reserved for use. The filtrates ①, ②, and ③ are combined, inactivated by high temperature, concentrated, and reserved for use, named Sample No. II.

[0047] 3. Separate honey extraction method Ginseng is crushed, sieved, and added with linden honey (Alpine Linden Honey produced by Jilin Aodong Health Technology Co., Ltd., production date: 20240129) at a solid-liquid ratio of 1:5, and kept at 85°C with ultrasonic treatment for 36 hours. The ginseng residue is filtered out, and the filtrate is reserved for use. The ginseng residue is extracted with deionized water twice at a solid-liquid ratio of 1:10, each time for 1 hour, filtered, and the combined filtrate is reserved for use, named Sample No. III.

[0048] 4. Separate enzymatic hydrolysis extraction method Ginseng, broken, sieved, enzymolyzed with an enzymolysis solution of food-grade lactase (β-galactosidase, derived from Bacillus subtilis) at a material-liquid ratio of 1:20 (prepared with 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 is 50 °C for three hours. After enzymolysis, filter, and reserve the filtrate. Extract the ginseng residue twice with deionized water at a material-liquid ratio of 1:10 for 1 hour each time, filter, and combine the filtrates for standby. Name it Sample No. IV.

[0049] II. Detection of Total Saponin Contents in Different Ginseng Extracts 1. Preparation of Reference Solution Take an appropriate amount of ginsenoside Re reference substance, weigh it accurately, and dissolve it in methanol to make a solution containing 1 mg per 1 mL, that is, obtain.

[0050] 2. Preparation of Test Samples Concentrate Sample No. I and Sample No. II to a small volume, extract twice with water-saturated n-butanol at a volume ratio of 1:3, recover the solvent, dry, and dissolve by ultrasonic treatment with methanol.

[0051] 3. Preparation of Standard Curve Precisely pipette 20 μL, 40 μL, 80 μL, 120 μL, 160 μL, and 200 μL of the reference solution into stoppered test tubes respectively. Evaporate the solvent at low temperature, add 0.5 mL of 1% vanillin perchloric acid test solution, mix well on a 60 °C constant temperature water bath, 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 removing the bubbles, use ultraviolet-visible spectrophotometry to measure the absorbance at a wavelength of 540 nm. Plot the standard curve with absorbance as the ordinate and concentration as the abscissa.

[0052] 4. Detection Results Detect the total saponin contents of Sample No. I and Sample No. II. The results are shown in Table 1. The total saponin content of Sample No. II is 1.45 times that of Sample No. I.

[0053] Table 1 Total Saponin Contents (n = 3) III. Detection of Monomeric Saponin Contents in Different Ginseng Extracts 1. Preparation of Mixed Reference Solution 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 a single reference substance solution. Take an appropriate amount of the single reference substance solution to prepare a mixed reference substance solution 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.

[0054] 2. Chromatographic conditions Chromatographic column: Agilent ZORBAX SB-Aq C18 chromatographic column (250 mm × 4.6 mm, 5 μm); Mobile phase: 0.1% phosphoric acid aqueous solution (A) - acetonitrile (B), the elution gradient is shown in Table 2 below; Flow rate: 1 mL / min; Column temperature: 35 °C; Detection wavelength: 203 nm; Injection volume: 10 μL.

[0055] Table 2 Elution program 3. Detection results Detect the monomer saponin contents of Sample No. I, Sample No. II, Sample No. III, and Sample No. IV. The results are shown in Table 3 and Table 4. The content of rare saponin in Sample No. II is 9.38 times that in Sample No. I, 5.93 times that in Sample No. III, and 2.38 times that in Sample No. IV.

[0056] Table 3 Partial rare saponin contents of Sample No. I and Sample No. II Table 4 Partial rare saponin contents of Sample No. III and Sample No. IV IV. Odor differences of different ginseng extracts Adopt electronic nose technology to detect flavor differences. Confirm that the surfaces of 18 metal oxide semiconductor sensors (MOS) are pollution-free, and gently wipe the sensor gas chamber with a special cleaning cotton swab. After turning on the equipment, preheat for 20 minutes to ensure the stability of the sensors (baseline drift should be < ±2%). Configure - connect the nitrogen carrier gas cylinder for the gas sampling system, and adjust the flow rate to 1 L / min. Collect for 60 s, clean for 2 min. Samples No. I and No. II are configured with the same crude drug concentration, 1 g / 10 mL. Add 3 mL of the sample to each sample bottle, let it stand for 4 hours, then conduct on-machine detection. Select the maximum odor value, load the data, select the top 8 sensors with the largest sensor differences, make a radar chart, and conduct PCA analysis.

[0057] The combined analysis of the electronic nose results and PCA results showed that there were significant differences in the odors of Samples No. I, No. II, and No. III. In particular, in terms of alcohol, ketone, and aldehyde components, such as components representing sweetness, fruitiness, and fragrance, Sample No. II was significantly stronger than Samples No. I and No. III.

[0058] V. Taste Differences of Different Ginseng Extracts The sensory evaluation method was carried out in accordance with the national standard "Plant Beverage" (GB / T 31326-2014). Samples No. I and No. II were prepared according to 1 g of ginseng raw material per 50 mL, sterilized, soaked in a transparent glass for 5 minutes, and stirred. Ten well-trained panel members (4 males and 6 females, aged 23-45 years) were invited to conduct sensory evaluations on the two ginseng beverages. The evaluations were scored from the aspects of appearance color, taste, ginseng flavor, and odor, with a scoring range of 0-100 and a total score of 4000 points, indicating the scores from the lowest to the highest.

[0059] Table 5 Sensory Evaluations of Different Ginseng Extracts The total score of Sample No. I was 2276 points, and that of Sample No. II was 3204 points. After processing, the flavor of ginseng extraction was effectively improved.

[0060] VI. Effects of Different Ginseng Extracts on Improving Sleep 1. Drosophila Sleep Monitoring System In this embodiment, the DAMS Drosophila activity monitoring system was used. Each Drosophila was placed in a single monitoring tube, and the detection tubes were successively inserted into the central position of the DAM2 Drosophila activity monitor. The monitor emits an infrared beam during monitoring. When a Drosophila moves in the tube, it blocks the infrared beam, and each block will be detected by the monitor and automatically counted. One monitor can simultaneously monitor the sleep activities of 32 Drosophila. During the experiment, the monitor was placed in a constant temperature and humidity incubator, set to a 12 h:12 h light-dark cycle, with a cultivation temperature of (25±1)°C and a relative humidity of 60%, and the sleep activities of Drosophila were recorded.

[0061] 2. Judgment Criteria for Drosophila Sleep The DAMS data acquisition software automatically records the number of times a Drosophila blocks the infrared beam every 5 minutes. If the number of activities of the Drosophila within these 5 minutes is zero, it is determined that the Drosophila is in a resting state, which meets the standard of sleep behavior, and it is considered that the Drosophila is in a sleep state, and its sleep time is recorded. Based on this, the average sleep time of Drosophila can be further calculated.

[0062] 3. Drosophila Sleep Deprivation Method Fix the DAM2 Drosophila activity monitor on the shaker, and set the shaking time and shaking interval. During shaking, the Drosophila in the monitoring tube cannot remain stationary as they vibrate with the detector, thus achieving mechanical sleep deprivation.

[0063] Experimental grouping: Control group (Control), add an equal amount of RO water to the basal medium, 32 flies; Model group / Sleep deprivation group (SD), add an equal amount of RO water to the basal medium, 32 flies; Sample Ⅰ (Ⅰ), prepare a medicated medium according to a drug dose of 1 mg / mL, 32 flies; Sample Ⅱ (Ⅱ), prepare a medicated medium according to a drug dose of 1 mg / mL, 32 flies.

[0064] 4. Drosophila sleep monitoring method Randomly divide female Drosophila into 4 groups. Place the Drosophila in the monitoring tube containing the medium, and insert the monitoring tube into the DAM2 Drosophila activity monitor in the middle, with 1 monitor for each group (n = 32). Except for the Control group, place the monitors of the other 3 groups on the shaker and shake for 5 s per minute. The Control group, SD group, and SD + drug treatment group are all placed in an environment with a 12 h:12 h light-dark cycle, and record the sleep situation of Drosophila for 72 h.

[0065] 5. Statistical analysis Use software for analysis. The data are analyzed by one-way ANOVA and t-test (comparison between two groups) and expressed as Means ± SD. p < 0.05 is considered to indicate a statistically significant difference. All experiments are repeated at least three times.

[0066] 6. Experimental results 6.1 Circadian rhythm of female Drosophila The circadian activities of Drosophila show a stress response to light conversion, with an activity peak after the light is turned on and after the light is turned off, indicating that this is the circadian rhythm activity of normal wild-type Drosophila. Compared with the model group, the circadian rhythm activities of female Drosophila fed with Sample Ⅰ and Sample Ⅱ are weakened, and the weakening of the circadian activity rhythm is more obvious in the Sample Ⅱ group.

[0067] 6.2 Sleep time of female Drosophila The results are as Figure 9 shown. After sleep deprivation, the sleep time of female Drosophila is significantly reduced. The total sleep time of female Drosophila fed with Sample Ⅰ and Sample Ⅱ is significantly increased, and the effect of feeding Sample Ⅱ is more significant (compared with the control group, #P < 0.05; compared with the model group, *P < 0.05, ***P < 0.001; compared between Sample Ⅰ and Sample Ⅱ, &P < 0.05).

[0068] 6.3 Sleep times of female Drosophila The results are as follows Figure 10 As shown, after sleep deprivation, the sleep times of female Drosophila melanogaster were significantly increased. When fed with Sample Ⅰ and Sample Ⅱ, the total sleep times of female Drosophila melanogaster were significantly reduced. This result reflects that both samples can effectively improve the sleep fragmentation of Drosophila melanogaster. And the Drosophila melanogaster fed with Sample Ⅱ showed a more significant improvement effect (compared with the control group, #P<0.05; compared with the model group, *P<0.05, ***P<0.001; compared with Sample Ⅰ, &P<0.05).

[0069] 6.4 Average sleep episode duration of female Drosophila melanogaster The results are as follows Figure 11 As shown, sleep deprivation significantly shortened the average sleep episode duration of female Drosophila melanogaster. For the female Drosophila melanogaster fed with Sample Ⅰ and Sample Ⅱ, their average sleep episode durations were significantly increased. This indicates that both samples can effectively improve the sleep quality of Drosophila melanogaster. The Drosophila melanogaster fed with Sample Ⅱ had a more significant improvement in sleep duration (compared with the control group, #P<0.05; compared with the model group, **P<0.01, ***P<0.001; compared with Sample Ⅰ, &P<0.05).

[0070] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions recorded in the foregoing embodiments, or perform equivalent replacements on some of the technical features. And these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A preparation method of ginseng extract containing protopanaxatriol, characterized in that, It includes the following steps: (S1) Prepare ginseng, crush it and sieve to obtain ginseng powder. Mix the ginseng powder with a honey solution and extract at 50 - 90 °C for 12 - 48 h to obtain a first extract and first ginseng residue; (S2) Mix the first ginseng residue and a β-galactosidase solution and extract at 37 - 50 °C for 1 - 10 h to obtain a second extract and second ginseng residue; (S3) Mix the second ginseng residue and water and perform water extraction at 80 - 100 °C for 0.5 - 5 h to obtain a third extract and third ginseng residue, and combine the first, second and third extracts.

2. The preparation method of the ginseng extract containing protopanaxatriol according to claim 1, characterized in that, In step (S1), the material-liquid ratio of the ginseng powder and the honey solution is 1:(1 - 20).

3. The preparation method of the ginseng extract containing protopanaxatriol according to claim 1, characterized in that, In step (S2), the material-liquid ratio of the first ginseng residue and the β-galactosidase solution is 1:(10 - 40).

4. The preparation method of the ginseng extract containing protopanaxatriol according to claim 1, characterized in that, In step (S2), the β-galactosidase solution includes β-galactosidase.

5. The preparation method of the ginseng extract containing protopanaxatriol according to claim 1, characterized in that, In step (S3), the material-liquid ratio of the second ginseng residue and water is 1:(5 - 30).

6. The preparation method of the ginseng extract containing protopanaxatriol according to claim 1, characterized in that, It further includes repeating the water extraction in step (S3).

7. Ginseng extract, characterized in that, It is obtained according to the preparation method described in any one of claims 1 - 6.

8. A pharmaceutical composition, characterized in that, It includes the ginseng extract described in claim 7 and a pharmaceutically acceptable carrier.

9. Use of the ginseng extract described in claim 7 in the preparation of a product for improving sleep.

10. The application according to claim 9, characterized in that, The product includes drugs and health products.

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