Polygala tenuifolia active ingredient co-extract and its preparation and application

Abstract

The present invention provides a co-extract of active ingredients from Polygala tenuifolia, and its preparation and application. Specifically, the co-extract of active ingredients from Polygala tenuifolia includes Polygala tenuifolia saponins, Polygala tenuifolia ketone III, 3,6'-dieserucyl sucrose, and 3,4,5-trimethoxycinnamic acid. The preparation method of the co-extract can simultaneously extract both water-soluble and alcohol-soluble active ingredients while also ensuring the conversion of Polygala tenuifolia saponins into Polygala tenuifolia saponins, thereby avoiding the loss of other active ingredients. The co-extract of active ingredients from Polygala tenuifolia can significantly improve the sleep quality of mice, with superior effects compared to existing Polygala tenuifolia powder and other single active ingredients, such as Polygala tenuifolia saponin extracts or Polygala tenuifolia ketone III extracts.

Description

Technical Field

[0001] The invention belongs to the field of phytochemistry, and particularly relates to a co-extract of active ingredients of Polygala tenuifolia and a preparation method and application thereof. Background Art

[0002] Polygala tenuifolia Willd., a plant of the genus Polygala in the family Polygalaceae, is used medicinally for its roots. It grows on sunny hillsides or along roadsides and is primarily distributed in Northeast, North, and Northwest China, as well as in Shandong, Anhui, Jiangxi, and Jiangsu. The tube of Polygala tenuifolia is hollow, cylindrical, and irregular, measuring 3 to 12 cm long and 0.3 to 1 cm in diameter. Its surface is gray or grayish-yellow, with dense, deep transverse wrinkles. Its cross-section is relatively flat, yellowish-white, and has a faint grassy aroma. Its taste is bitter and slightly pungent, with a tingling throat. Polygala tenuifolia has calming, intellectual, expectorant, and swelling-reducing properties. It is used to treat insomnia, dreaminess, forgetfulness, palpitations, confusion, persistent expectoration, sores, swelling, and breast pain caused by heart-kidney disharmony.

[0003] The hallmark active ingredients of Polygala root extracts include triterpenoid saponins, ketones, oligosaccharides, and other active ingredients. Polygala root saponins, one of the main active ingredients of Polygala root, are all pentacyclic triterpenoid saponins of the oleanolic acid type. They exhibit anti-inflammatory, antioxidant, anti-aging, neuroprotective, intellectual, and antidepressant properties. The structural nucleus of these saponins is characterized by a hydroxyl group often substituted at the C-2 position, unsaturated double bonds at C-12 and C-13, and disaccharide chains, with glucose attached at C-3 and an ester bond to sucrose at C-28. These saponins are generally hemolytic, but alkaline hydrolysis of the C-28 ester bond eliminates the hemolytic effect and significantly reduces toxicity. Conversion of Polygala root saponins to Polygala tenuifolia saponins eliminates the toxic effects of these saponins. Furthermore, Polygala tenuifolia saponins exhibit strong chemical stability and potent anticholinesterase activity, making them significantly superior to Polygala root saponins in improving neurological function. Xanthone, also known as benzochromone, has diuretic, antibacterial, anticancer, and antidepressant activities. Over 80 xanthone compounds have been isolated from Polygala plants, primarily existing in the form of simple xanthone compounds and xanthone glycosides. Oligosaccharide esters found in Polygala plants primarily use sucrose as a common nucleus, with glucose (a small percentage is rhamnose) linked to it via various glycosidic bonds, such as dierucyl sucrose. These compounds have antidepressant, neuroprotective, neuronal regeneration, and memory-enhancing properties. Other components, such as 3,4,5-trimethoxycinnamic acid and methyl 3,4,5-trimethoxycinnamate, exhibit sedative and hypnotic activity. Polygala tenuifolia saponins, which contain 3,4,5-trimethoxycinnamic acid, are products of the conversion of Polygala tenuifolia saponins to Polygala tenuifolia saponins.

[0004] However, conventional methods of boiling Polygala root fail to fully capture its active ingredients, and the active ingredients consumed only represent a small fraction of the root itself. Direct consumption of Polygala root powder is also a common method, but absorption efficiency is low, and a large amount of Polygala root saponins are not converted into Polygala tenuifolia saponins, resulting in high toxicity and poor efficacy. Consequently, reports have used alkaline hydrolysis to extract Polygala tenuifolia saponins, or alcohol to extract Polygala tenuifolia ketone III and oligosaccharide lipids. However, neither alkaline hydrolysis nor alcohol extraction can simultaneously obtain Polygala tenuifolia saponins, Polygala tenuifolia ketone III, and oligosaccharide lipids. High-concentration alkali treatment easily leads to the instability of substances such as Polygala tenuifolia ketone III, oligosaccharide lipids, and 3,4,5-trimethoxycinnamic acid, a conversion product of Polygala tenuifolia saponins. Therefore, in the currently available data, there are few reports on methods for the co-extraction of active substances such as Polygala tenuifolia saponins, Polygala tenuifolia ketone III, 3,6'-dicerinoylsucrose, and 3,4,5-trimethoxycinnamic acid. Summary of the Invention

[0005] The present invention provides a co-extract of polygala tenuifolia active ingredients comprising multiple active ingredients such as polygala tenuifolia saponins, polygala tenuifolia ketone III, 3,6'-diesinapoylsucrose and 3,4,5-trimethoxycinnamic acid, as well as preparation and application thereof. The preparation method ensures a high conversion rate of polygala tenuifolia saponins while ensuring the stability of polygala tenuifolia ketone III, 3,6'-diesinapoylsucrose and 3,4,5-trimethoxycinnamic acid.

[0006] In a first aspect of the present invention, a co-extract of active ingredients of Polygala tenuifolia is provided, wherein the co-extract of active ingredients of Polygala tenuifolia comprises active ingredients selected from the group consisting of Polygala tenuifolia saponins, Polygala tenuifolia ketone III, 3,6'-diesinapoylsucrose and 3,4,5-trimethoxycinnamic acid.

[0007] In another preferred embodiment, the polygala tenuifolia saponin content in the co-extract of the polygala tenuifolia active ingredients is ≥0.10 mg / mL.

[0008] In another preferred embodiment, the content of Polygala tenuifolia saponin in the co-extract of active ingredients of Polygala tenuifolia is ≥0.14 mg / mL; preferably, ≥0.16 mg / mL.

[0009] In another preferred embodiment, the co-extract of the active ingredients of Polygala tenuifolia further has one or more characteristics selected from the following group:

[0010] (i) 3,6'-diesinapoylsucrose content ≥ 0.05 mg / mL;

[0011] (ii) 3,4,5-trimethoxycinnamic acid content ≥ 0.01 mg / mL;

[0012] (ii) The content of polygala tenuifolia ketone III is ≥ 0.02 mg / mL.

[0013] In another preferred embodiment, the content of 3,6'-dieserucylsucrose in the co-extract of the active ingredients of Polygala tenuifolia is ≥0.07 mg / mL; preferably, ≥0.08 mg / mL.

[0014] In another preferred embodiment, the content of 3,4,5-trimethoxycinnamic acid in the co-extract of the active ingredients of Polygala tenuifolia is ≥0.018 mg / mL; preferably, ≥0.020 mg / mL.

[0015] In another preferred embodiment, the content of polygala tenuifolia ketone III in the co-extract of the active ingredients of polygala tenuifolia is ≥0.028 mg / mL; preferably, ≥0.03 mg / mL.

[0016] In another preferred embodiment, the content of total saponins in the co-extract of active ingredients of Polygala tenuifolia is ≥1 mg / mL; preferably, ≥1.2 mg / mL.

[0017] In another preferred embodiment, the co-extract of the active ingredients of Polygala tenuifolia is prepared by the following method:

[0018] (1) Providing raw materials of Polygala tenuifolia, drying and crushing them to obtain Polygala tenuifolia powder;

[0019] (2) Take the Polygala powder obtained in step (1), extract it with water to obtain the Polygala water extract, filter it, and collect the supernatant and solid part respectively:

[0020] (3) Take the supernatant obtained in step (2), add alkaline solution to react, filter, and obtain alkaline-treated filtrate (first filtrate) and filter cake:

[0021] (4) Take the filter cake obtained in step (3), dry and crush it, add 70-98 vv% ethanol to extract it, and filter it to obtain the alcohol extraction supernatant (second filtrate) and the filter cake:

[0022] (5) The alcohol extraction supernatant (second filtrate) obtained in step (4) and the alkali-treated filtrate (first filtrate) obtained in step (3) are combined to obtain a co-extract of the active ingredients of Polygala tenuifolia.

[0023] In a second aspect of the present invention, a method for preparing the co-extract of the active ingredients of Polygala tenuifolia according to the first aspect of the present invention is provided, the method comprising:

[0024] (1) Providing raw materials of Polygala tenuifolia, drying and crushing them to obtain Polygala tenuifolia powder;

[0025] (2) Take the Polygala powder obtained in step (1), extract it with water to obtain the Polygala water extract, filter it, and collect the supernatant and solid part respectively:

[0026] (3) Take the supernatant obtained in step (2), add alkaline solution to react, filter, and obtain alkaline-treated filtrate (first filtrate) and filter cake:

[0027] (4) Take the filter cake obtained in step (3), dry and crush it, add 70-98 vv% ethanol to extract it, and filter it to obtain the alcohol extraction supernatant (second filtrate) and the filter cake:

[0028] (5) The alcohol extraction supernatant (second filtrate) obtained in step (4) and the alkali-treated filtrate (first filtrate) obtained in step (3) are combined to obtain a co-extract of the active ingredients of Polygala tenuifolia.

[0029] In another preferred embodiment, the step (5) further comprises:

[0030] The alcohol extraction supernatant (second filtrate) obtained in step (4) and the alkali-treated filtrate (first filtrate) obtained in step (3) are mixed and diluted with water to obtain a dilution, and 2-10 wt% maltodextrin is added to the dilution, and the mixture is spray-dried to obtain a co-extract of the active ingredients of Polygala tenuifolia.

[0031] In another preferred embodiment, the spray drying conditions are: inlet air temperature is between 135 and 170° C., feed pressure is between 0.2 and 0.4 bar, and feed speed is between 30 and 50 rpm / min.

[0032] In another preferred embodiment, after dilution with water, the ethanol concentration in the dilution liquid is ≤25wt%, preferably, the ethanol concentration in the dilution liquid is ≤20wt%.

[0033] In another preferred embodiment, the drying temperature in step (4) is 55-65°C.

[0034] In another preferred embodiment, the concentration of ethanol in step (4) is 90-98 vv%.

[0035] In another preferred embodiment, the ratio of ethanol addition in step (4) is (4-18) mL ethanol: 1 g raw material; preferably (8-12) mL ethanol: 1 g raw material.

[0036] In another preferred embodiment, the ethanol extraction temperature in step (4) is 0-28°C; preferably, 15-25°C.

[0037] In another preferred embodiment, the concentration of the alkaline solution in step (3) is 0.01-0.5 wt%.

[0038] In another preferred embodiment, the concentration of the alkaline solution in step (3) is 0.1-0.2 wt%.

[0039] In another preferred embodiment, the alkaline solution in step (3) is sodium hydroxide.

[0040] In another preferred embodiment, the pH of the solution after adding the alkaline solution in step (3) is 8-12.

[0041] In another preferred embodiment, the reaction temperature in step (3) is 100-150°C; preferably 110-120°C.

[0042] In another preferred embodiment, the reaction time in step (3) is 0.2-5 h; preferably 0.5-1.5 h.

[0043] In another preferred embodiment, step (3) further comprises: adding citric acid to adjust the pH of the solution to about 5-8, preferably, pH=6.

[0044] In another preferred embodiment, the raw material in step (1) is cored Polygala tube.

[0045] In another preferred embodiment, step (1) further comprises ultrasonic cleaning of the raw material:

[0046] In another preferred embodiment, step (1) further comprises: soaking in 1 kg / (8-12) L water and performing ultrasonic cleaning for 1 to 10 minutes.

[0047] In another preferred embodiment, the drying temperature in step (1) is 55-65°C.

[0048] In another preferred embodiment, the ratio of water added in step (1) is (8-12) L: 1 kg of raw material.

[0049] In another preferred embodiment, the water extraction temperature in step (1) is 0-28°C, preferably 2-15°C.

[0050] In another preferred embodiment, the water extraction time in step (1) is 2 to 24 hours, preferably 10 to 15 hours.

[0051] In the third aspect of the present invention, there is provided a use of the co-extract of the active ingredients of Polygala tenuifolia as described in the first aspect of the present invention, for preparing a composition for improving sleep.

[0052] In another preferred embodiment, the composition has one or more characteristics selected from the following group:

[0053] (a) shortening of the barbiturate latency to sleep;

[0054] (b) Prolonged barbiturate sleep.

[0055] In another preferred embodiment, the co-extract of the active ingredients of Polygala tenuifolia has the effect of improving sleep.

[0056] In another preferred embodiment, the co-extract of the active ingredients of Polygala tenuifolia has the effect of shortening the barbiturate latency period and / or prolonging the barbiturate sleep time.

[0057] In a fourth aspect of the present invention, a composition is provided, comprising: (1) the co-extract of the active ingredients of Polygala tenuifolia as described in the first aspect of the present invention; and (2) additional ingredients that can be used in food or medicine.

[0058] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features described in detail below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be listed here one by one. BRIEF DESCRIPTION OF THE DRAWINGS

[0059] Figure 1 The experimental results of various Polygala extracts shortening the sleep latency period of sodium pentobarbital are presented. Figure 1 The picture above shows the number of sleeping animals. Figure 1 The picture below shows the time to fall asleep.

[0060] Figure 2 These are the experimental results of various Polygala tenuifolia extracts on prolonging the sleeping time after sodium pentobarbital administration. DETAILED DESCRIPTION

[0061] After extensive and in-depth research, the inventors unexpectedly developed a co-extract of Polygala root active ingredients that contains multiple active ingredients from Polygala root and has a significant effect on improving sleep. The co-extract contains four key active ingredients from Polygala root: Polygala tenuifolia saponins, Polygala tenuifolia ketone III, 3,6-dienasoyl sucrose, and 3,4,5-trimethoxycinnamic acid. The preparation method for the co-extract includes water extraction and ethanol extraction, ensuring that both the water-soluble active substances (Polygala tenuifolia saponins) and the water-insoluble substances (Polygala tenuifolia ketone III and 3,6'-dienasoyl sucrose) from Polygala root can be extracted. The preparation method also uses a low concentration of alkali, making it safe and low-pollution. Based on this, the present invention was completed.

[0062] The main advantages of the present invention are:

[0063] 1. Superior activity: The co-extract of active ingredients from Polygala tenuifolia L. described in this invention includes four key active ingredients from Polygala tenuifolia L.: polygala tenuifolia saponins, polygala tenuifolia ketone III, 3,6-diesinapoylsucrose, and 3,4,5-trimethoxycinnamic acid. Animal experiments have shown that it is most effective in improving sleep quality in mice.

[0064] 2. High content of active ingredients and minimal degradation: The method described in the present invention includes water extraction and ethanol extraction, ensuring that both water-soluble active substances (polygala saponins) and water-insoluble substances (polygala tenuifolia saponins) can be extracted. Unlike the reported method for extracting polygala tenuifolia saponins (which directly adds ethanol after alkaline hydrolysis), this process separates the water extract and solid residue before alkaline treatment, and the solid residue is extracted with ethanol. Tests have shown that this method can avoid the hydrolysis of active substances such as polygala tenuifolia saponins and 3,6'-dienasoylsucrose under alkaline conditions.

[0065] 3. Safe and Effective Preparation Method: The method described in this invention utilizes a low alkali treatment concentration (preferably 0.15 wt% sodium hydroxide), a pH of approximately 10, and a treatment temperature of 100-115°C, unlike existing techniques that require high alkali concentrations. This method requires minimal industrial facilities and poses minimal potential risks to operators. It also ensures a higher conversion rate of Polygala tenuifolia saponins and maximizes the stability of other active ingredients, such as 3,4,5-trimethoxycinnamic acid.

[0066] 4. Unexpected Effects of Drying: The present inventors unexpectedly discovered that drying the solid portion of Polygala root extract at 60°C for more than 3 hours, followed by extraction with 75% or higher ethanol, significantly increases the extraction yield of Polygala root linalool III and 3,6'-dicerinoylsucrose. Comparative experiments also revealed that drying the water extract not only reduces moisture content and increases the yield of active ingredients, but also facilitates the release of 3,6'-dicerinoylsucrose and Polygala root linalool III, further improving yield. Furthermore, the ethanol soaking process eliminates the complex heating and reflux process, simplifying the method.

[0067] the term

[0068] In order to make the present invention easier to understand, certain technical and scientific terms are specifically defined below. Unless otherwise clearly defined in this article, all other technical and scientific terms used herein have the meanings generally understood by those of ordinary skill in the art to which the present invention belongs. Before describing the present invention, it should be understood that the present invention is not limited to the specific methods and experimental conditions described, because such methods and conditions can change. It should also be understood that the terms used herein are intended only to describe specific embodiments and are not intended to be restrictive, and the scope of the present invention will be limited only by the appended claims.

[0069] Unless defined otherwise, 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 belongs.

[0070] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention and are not intended to limit the scope of the invention. Unless otherwise indicated, percentages and parts are weight percentages and weight fractions.

[0071] General detection methods

[0072] Total saponins detection method

[0073] (1) Principle

[0074] The total saponins from Polygala tenuifolia reacted with vanillin under acidic conditions to develop color (measured at 560 nm), and the content was calculated using ginsenoside Re as the reference.

[0075] (2) Reagents and equipment

[0076] Color developer: 0.5% vanillin in glacial acetic acid (0.5 g → 100 mL glacial acetic acid)

[0077] Reaction acid: perchloric acid (analytical grade)

[0078] Standard ginsenoside Re (purity ≥98%) (C 48 H 82 O 18 , CAS No.: 257-814-6)

[0079] Solvents: methanol, anhydrous ethanol, glacial acetic acid

[0080] Equipment: UV spectrophotometer, constant temperature water bath

[0081] (3) Solution preparation

[0082] Preparation of standard stock solution: Accurately weigh 10 mg of ginsenoside Re standard to the nearest 0.00001 g, prepare a 1 mg / mL standard stock solution with methanol, and store in a 4°C refrigerator for 6 months.

[0083] Vanillin solution: Dissolve 0.5 g of vanillin (C8H8O3) in glacial acetic acid and dilute to 100 mL.

[0084] (4) Analysis

[0085] Standard working solution: Take 0µL, 20µL, 40µL, 60µL, 80µL, and 100µL of the standard stock solution (equivalent to 0µg, 20µg, 40µg, 60µg, 80µg, and 100µg), respectively, and place them in 10mL colorimetric tubes. Evaporate them to dryness in a 60℃ water bath and set aside.

[0086] Preparation of test solution: Take 10~50µl of the test solution, place it in a centrifuge tube, heat it in a 60℃ constant temperature water bath to evaporate, and set aside.

[0087] Plotting the standard working curve: add 0.2 ml of vanillin solution to the standard working solution, mix well to dissolve the residue, then add 0.8 ml of perchloric acid, mix well, heat in a 60°C constant temperature water bath for 10 min, take out, cool in an ice bath, add glacial acetic acid to make the volume up to 5.0 mL, shake well, and perform colorimetry at a wavelength of 560 nm using a 1 cm cuvette to obtain the absorbance value. Plot the standard curve with concentration as the horizontal axis and absorbance as the vertical axis.

[0088] (5) Determination

[0089] In the test tube, refer to the "Standard Working Curve Drawing" in step (4) to obtain the absorbance value, and calculate the content of Polygala tenuifolia saponins in the sample solution to be tested using the standard curve.

[0090] Blank test: Perform the test according to the test procedures except that no sample is added.

[0091] (6) Calculation of results:

[0092]

[0093] Where:

[0094] X — total saponin content of Polygala tenuifolia in the sample, in grams per 100 grams (g / 100 g or g / 100 mL);

[0095] A —the mass of total saponins in the test solution calculated from the standard curve, in micrograms (µg);

[0096] V1—total volume of sample preparation, in milliliters (mL);

[0097] V2—volume of sample preparation solution for determination, in milliliters (mL);

[0098] m—sample mass or sample volume, in grams or milliliters (g or mL).

[0099] The calculation result should be rounded to two significant figures.

[0100] Detection method of saponins in Polygala tenuifolia

[0101] (1) Reference documents: Determined in accordance with the "Content Determination of Medicinal Materials and Medicinal Pieces of Polygala tenuifolia" in Volume 1 of the Chinese Pharmacopoeia and "General Chapter 0512 High Performance Liquid Chromatography" in Volume 4 of the Chinese Pharmacopoeia.

[0102] (2) Reagents and equipment

[0103] Standard Polygala tenuifolia saponin standard (C 36 H 56 O 12, CAS No.: 20183-47-5 Purity ≥98%)

[0104] Reagents: methanol, phosphoric acid (chromatographic grade); Wahaha purified water

[0105] Equipment: Shimadzu LC-2030 HPLC, ECLIPSE PLUS C18 (3.5 μm, 4.6 × 100 mm)

[0106] (3) Solution preparation

[0107] Preparation of 1 mg / mL standard stock solution: Accurately weigh 10 mg of the Polygala tenuifolia saponin standard to the nearest 0.00001 g, add methanol to prepare a 1 mg / mL standard stock solution, and store in a 4°C refrigerator for 6 months.

[0108] Preparation of a series of reference solutions: Place 0µL, 50µL, 100µL, 200µL, 400µL, and 800µL of the standard stock solution into EP tubes, respectively. Add methanol to 1mL to create 0.0mg / mL, 0.05mg / mL, 0.1mg / mL, 0.2mg / mL, 0.4mg / mL, and 0.8mg / mL solutions of Polygala tenuifolia saponins. Filter all reference solutions through a 0.45µm filter.

[0109] Preparation of test solution: Take 1 mL of the test sample and filter it through a 0.45 μm filter membrane.

[0110] (4) Chromatographic conditions

[0111] The mobile phase was methanol-0.05% phosphoric acid solution (70:30); the flow rate was 1.0 mL / min; the detection wavelength was 210 nm; the chromatographic column was ECLIPSE PLUS C18 (3.5 μm, 4.6×100 mm); the column temperature was 30°C; and the acquisition time was 30 minutes.

[0112] (5) Analysis

[0113] Accurately measure 10 μL of the reference solution and the test solution respectively, inject them into the liquid chromatograph, and record the chromatogram.

[0114] (6) Data processing

[0115] The raw data generated by the high performance liquid chromatograph are automatically integrated and peak marked by the instrument's own software.

[0116] (7) Result processing and calculation

[0117] Perform a linear regression on the measured peak areas using various concentrations of the Polygala tenuifolia saponin reference solution (mg / mL) to determine the linear regression equation Y = bx + a. The linear regression correlation coefficient, r, should be no less than 0.99.

[0118] Substitute the measured peak area of ​​the test sample into the linear regression equation to calculate the content of Polygala tenuifolia saponins in the test sample (mg / mL).

[0119] Detection method of 3,6'-dierucyl sucrose

[0120] (1) Reference documents: Determined in accordance with the "Content Determination of Medicinal Materials and Medicinal Pieces of Polygala tenuifolia" in Volume 1 of the Chinese Pharmacopoeia and "General Chapter 0512 High Performance Liquid Chromatography" in Volume 4 of the Chinese Pharmacopoeia.

[0121] (2) Reagents and equipment

[0122] Standard 3,6'-diesinapoylsucrose standard (C 34 H 42 O 19 , CAS No.: 139891-98-8, purity ≥98%)

[0123] Reagents: acetonitrile, phosphoric acid (chromatographic grade); Wahaha purified water

[0124] Equipment: Shimadzu LC-2030 HPLC, ECLIPSE PLUS C18 (3.5 μm, 4.6 × 100 mm)

[0125] (3) Solution preparation

[0126] Preparation of 1 mg / mL standard stock solution: Accurately weigh 10 mg of 3,6'-diesinapoylsucrose standard to the nearest 0.00001 g, add methanol to prepare a 1 mg / mL standard stock solution, and store in a 4°C refrigerator for 6 months.

[0127] Preparation of a series of reference solutions: Place 0µL, 50µL, 100µL, 200µL, 400µL, and 800µL of the standard stock solution into EP tubes, respectively. Add methanol to 1mL to obtain 0mg / mL, 0.050mg / mL, 0.100mg / mL, 0.200mg / mL, 0.400mg / mL, and 0.800mg / mL 3,6'-diesinapoylsucrose solutions. Filter all reference solutions through a 0.45µm filter.

[0128] Preparation of test solution: Take 1 mL of the test sample and filter it through a 0.45 μm filter membrane.

[0129] (4) Chromatographic conditions

[0130] The mobile phase was acetonitrile-0.05% phosphoric acid solution (18:82); the flow rate was 1.0 mL / min; the detection wavelength was 320 nm; the chromatographic column was ECLIPSE PLUS C18 (3.5 μm, 4.6×100 mm); the column temperature was 30°C; and the acquisition time was 35 minutes.

[0131] (5) Analysis

[0132] Accurately measure 10 μl of the reference solution and the test solution respectively, inject them into the liquid chromatograph, and record the chromatogram.

[0133] (6) Data processing

[0134] The raw data generated by the high performance liquid chromatograph are automatically integrated and peak marked by the instrument's own software.

[0135] (7) Result processing and calculation

[0136] Perform a linear regression on the measured peak areas using various concentrations of 3,6'-diesinapoylsucrose reference solution (mg / mL) to determine the linear regression equation Y = bx + a. The linear regression correlation coefficient, r, should be no less than 0.99.

[0137] Substitute the measured peak area of ​​the test sample into the linear regression equation to calculate the 3,6'-diesinapoylsucrose content (mg / mL) in the test sample.

[0138] 3,4,5-Trimethoxycinnamic acid content detection method

[0139] (1) References: Zhao Mengjun, Li Chenchen, Gan Long, et al. Determination of the contents of eight components of Polygala tenuifolia by alkali hydrolysis by HPLC [J]. Modern Chinese Medicine, 2020, 22(3)

[0140] Standard 3,4,5-trimethoxycinnamic acid standard (C 12 H 14 O5, CAS number: 90-50-6, purity ≥98%)

[0141] Reagents: acetonitrile, phosphoric acid (chromatographic grade); Wahaha purified water

[0142] Equipment: Shimadzu LC-2030 HPLC, ECLIPSE PLUS C18 (3.5 μm, 4.6 × 100 mm)

[0143] (3) Solution preparation:

[0144] Preparation of 1 mg / mL standard stock solution: Accurately weigh 10 mg of 3,4,5-trimethoxycinnamic acid standard to the nearest 0.00001 g, add methanol to make a 1 mg / mL standard stock solution, and store in a 4°C refrigerator for 6 months.

[0145] Preparation of a series of reference solutions: Place 0µL, 20µL, 40µL, 80µL, 160µL, and 320µL of the standard stock solution into EP tubes, respectively. Add methanol to 1mL to create 0.0mg / mL, 20µg / mL, 40µg / mL, 80µg / mL, 160µg / mL, and 320µg / mL 3,4,5-trimethoxycinnamic acid solutions. Filter all reference solutions through a 0.45µm filter.

[0146] Preparation of test solution: Take 1 ml of the test sample and filter it through a 0.45 μm filter membrane.

[0147] (4) Chromatographic conditions

[0148] The mobile phase was acetonitrile (A)-0.05% phosphoric acid-water solution (B), gradient elution (0-5 min, 25%A; 6-25 min, 25%-45%A; 26-30 min, 45%-80%A; 31-35 min, 80%A); the flow rate was 0.5 mL / min; the column temperature was 30°C; the detection wavelength was 310 nm; the chromatographic column was ECLIPSE PLUS C18 (3.5 μm, 4.6×100 mm); the column temperature was 30°C; and the acquisition time was 30 min.

[0149] (5) Analysis

[0150] Accurately measure 10 μL of the reference solution and the test solution respectively, inject them into the liquid chromatograph, and record the chromatogram.

[0151] (6) Data processing

[0152] The raw data generated by the high performance liquid chromatograph are automatically integrated and peak marked by the instrument's own software.

[0153] (7) Result processing and calculation

[0154] Perform a linear regression on the measured peak areas using various concentrations of 3,4,5-trimethoxycinnamic acid reference solutions (μg / mL) to determine the linear regression equation Y = bx + a. The linear regression correlation coefficient r should be no less than 0.99.

[0155] Substitute the measured peak area of ​​the test sample into the linear regression equation to calculate the content of 3,4,5-trimethoxycinnamic acid in the test sample (μg / mL).

[0156] Detection method of polygala tenuifolia ketone Ⅲ content

[0157] (1) Reference documents: Determined in accordance with the "Content Determination of Medicinal Materials and Medicinal Pieces of Polygala tenuifolia" in Volume 1 of the Chinese Pharmacopoeia and "General Chapter 0512 High Performance Liquid Chromatography" in Volume 4 of the Chinese Pharmacopoeia.

[0158] (2) Reagents and equipment

[0159] Standard Polygala tenuifolia ketone III standard (C 25 H 28 O 15 , CAS No.: 162857-78-5, purity ≥98%)

[0160] Reagents: methanol, phosphoric acid (chromatographic grade); Wahaha purified water

[0161] Equipment: Shimadzu LC-2030 HPLC, ECLIPSE PLUS C18 (3.5 μm, 4.6 × 100 mm)

[0162] (3) Solution preparation:

[0163] Preparation of 1 mg / mL standard stock solution: Accurately weigh 10 mg of polygala tenuifolia ketone III standard to the nearest 0.00001 g, add methanol to prepare a 1 mg / mL standard stock solution, and store in a 4°C refrigerator for 6 months.

[0164] Preparation of a series of reference solutions: Place 0µL, 20µL, 40µL, 80µL, 160µL, and 320µL of the standard stock solution into EP tubes, respectively. Add methanol to 1mL to obtain 0µg / mL, 20µg / mL, 40µg / mL, 80µg / mL, 160µg / mL, and 320µg / mL solutions of Polygala tenuifolia III. Filter all reference solutions through a 0.45µm filter.

[0165] Preparation of test solution: Take 1 mL of the test sample and filter it through a 0.45 μm filter membrane.

[0166] (4) Chromatographic conditions

[0167] The mobile phase was acetonitrile-0.05% phosphoric acid solution (18:82); the flow rate was 1.0 mL / min; the detection wavelength was 320 nm; the chromatographic column was ECLIPSE PLUS C18 (3.5 μm, 4.6×100 mm); the column temperature was 30°C; and the acquisition time was 35 minutes.

[0168] (5) Analysis

[0169] Accurately measure 10 μl of the reference solution and the test solution respectively, inject them into the liquid chromatograph, and record the chromatogram.

[0170] (6) Data processing

[0171] The raw data generated by the high performance liquid chromatograph are automatically integrated and peak marked by the instrument's own software.

[0172] (7) Result processing and calculation

[0173] Perform a linear regression of the measured peak areas using various concentrations of polygala tenuifolia ketone III reference solution (μg / mL) to determine the linear regression equation Y = bx + a. The linear regression correlation coefficient, r, should be no less than 0.99.

[0174] Substitute the measured peak area of ​​the test sample into the linear regression equation to calculate the content of polygala tenuifolia ketone III in the test sample (μg / mL).

[0175] Example 1: Preparation of co-extracts of active ingredients from Polygala tenuifolia

[0176] A method for selecting an extraction process for Polygala tenuifolia is provided, wherein the specific steps are as follows:

[0177] Step 1: Take 20g of dried Polygala tube and grind it with a grinder, then sieve it through a 100-mesh sieve to obtain Polygala powder. Divide the powder into three groups, 5g each.

[0178] Step 2: Take 5g of the Polygala tenuifolia powder obtained in Step 1 above and soak it in 50mL of water overnight. Centrifuge to separate the supernatant and solid precipitate. Add 1% sodium hydroxide to the supernatant, boil at 100°C for 1 hour, and filter through a 100-mesh sieve to obtain an aqueous extract. Add 50mL of anhydrous ethanol to the solid precipitate, soak it overnight, and filter through filter paper to obtain an alcohol extract.

[0179] Step 3: Combine the aqueous and alcohol extracts, add 5% maltodextrin, and spray dry using a spray dryer. Spray drying conditions were: inlet air temperature 140°C, feed pressure 0.2 bar, and feed rate 30 rpm / min. Collect approximately 5 g of the resulting powder, obtaining the sample from Group E1.

[0180] Step 4: Take 0.1 g of powder (1 / 50 of the total amount), dissolve it in 1 mL of methanol, and test the contents of total saponins, Polygala tenuifolia saponins, and 3,6'-diesinapoylsucrose, respectively. The specific test results are shown in Table 1.

[0181] Comparative Example C1: Preparation and extraction method of common Polygala tenuifolia saponins

[0182] 5 g of the Polygala tenuifolia powder obtained in step 1 of Example 1 was taken and soaked overnight in 50 mL of water. The supernatant was collected by centrifugation, and 10% sodium hydroxide was added to the supernatant for 2 h. The pH was adjusted to 4.0 with concentrated hydrochloric acid. The solution was concentrated to 5 mL, 45 mL of anhydrous ethanol was added, and the solution was filtered with filter paper to obtain a filtrate.

[0183] The filtrate was diluted 2-fold with water, 5% maltodextrin was added, and the mixture was spray-dried using a spray dryer. Spray-drying conditions were: inlet air temperature of 140°C, feed pressure of 0.2 bar, and feed rate of 30 rpm / min. Approximately 5 g of the resulting powder was collected. This yielded the sample from Group C1.

[0184] 0.1 g of powder (1 / 50 of the total amount) was taken and dissolved in 1 mL of methanol. The contents of total saponins, Polygala tenuifolia saponins and 3,6'-diesinapoylsucrose were tested respectively. The specific test results are shown in Table 1.

[0185] Comparative Example C2 Organic Phase Reflux Extraction Preparation and Extraction Method

[0186] 5 g of the Polygala root powder obtained in step 1 of Example 1 was extracted using organic phase reflux extraction. 50 ml of 90% ethanol was added to the extract, which was placed in an extraction vessel. The extract was heated to reflux and maintained at reflux for 1.5 hours. Reflux extraction was performed three times. The extracts after reflux extraction were combined, cooled, and supplemented with 90% ethanol to make up the loss. The mixture was shaken well and filtered through filter paper to obtain a filtrate.

[0187] The filtrate was diluted 2-fold with water, 5% maltodextrin was added, and the mixture was spray-dried using a spray dryer. Spray-drying conditions were: inlet air temperature of 140°C, feed pressure of 0.2 bar, and feed rate of 30 rpm / min. Approximately 5 g of the resulting powder was collected. This yielded the sample from Group C2.

[0188] 0.1 g of powder (1 / 50 of the total amount) was taken and dissolved in 1 mL of methanol. The contents of total saponins, Polygala tenuifolia saponins and 3,6'-diesinapoylsucrose were tested respectively. The specific test results are shown in Table 1.

[0189] Table 1: Content of substances extracted from different Polygala root extracts

[0190]

[0191] Conclusion: In comparative example C1, i.e., the method for preparing and extracting Polygala tenuifolia saponins, it can be seen that the content of Polygala tenuifolia saponins in the extract is high, which may be due to the high concentration of sodium hydroxide. Therefore, a lot of Polygala tenuifolia saponins were converted, but no 3,6'-dicorinoylsucrose was detected, proving that it is impossible to effectively extract 3,6'-dicorinoylsucrose and other effective components with sedative and sleep-inducing effects; in comparative example C2, i.e., the organic phase reflux extraction, it can be observed that the total saponin content in the extract is the highest, but no Polygala tenuifolia saponins were detected, proving that most of them are ordinary Polygala tenuifolia saponins and almost no Polygala tenuifolia saponins; and in the extract obtained by Example 1, i.e., the mild Polygala tenuifolia conversion and extraction method described in the present application, two active ingredients, i.e., Polygala tenuifolia saponins and 3,6'-dicorinoylsucrose, were detected at the same time, and the contents of both were high. The two active ingredients can be co-extracted. When Polygala tenuifolia saponins are converted into Polygala tenuifolia saponins, 3,6'-dicorinoylsucrose and other effective components with sedative and sleep-inducing effects can be extracted at the same time.

[0192] Example 2: Optimization of Polygala tenuifolia Water Extraction and Alkali Treatment Process

[0193] An optimization scheme for the water extraction and alkali treatment process of Polygala tenuifolia, the specific steps are as follows:

[0194] Step 1: Grind 20 g of dried Polygala tubes using a grinder and sieve through a 100-mesh sieve to obtain Polygala powder. Add the powder to 200 mL of water and soak overnight. Centrifuge and collect the supernatant.

[0195] Step 2: Take 50 mL of the above supernatant, add 0.05 wt% sodium hydroxide, dissolve, and divide into 6 small portions. Treat the 6 small portions according to the conditions in Table 2, and add citric acid to adjust the pH to 6.0.

[0196] Table 2: Sample processing reaction conditions

[0197]

[0198] Step 3: Take 50 ml of the above supernatant, add 0.15 wt% sodium hydroxide, dissolve, and divide into 6 small portions. Treat the 6 small portions according to the conditions in Table 2, and add citric acid to adjust the pH to 6.0.

[0199] Step 4: Take 50 ml of the above supernatant, add 0.5 wt% sodium hydroxide, dissolve, and divide into 6 small portions. Treat the 6 small portions according to the conditions in Table 2, and add citric acid to adjust the pH to 6.0.

[0200] Step 5. The 18 treated samples were tested for the contents of Polygala tenuifolia saponins and 3,4,5-trimethoxycinnamic acid, and the test results are shown in Table 3.

[0201] Table 3: Alkali treatment process optimization data

[0202]

[0203] The experimental results are shown in Table 3, from which we can see that:

[0204] Under the same conditions, when the alkali treatment concentration increased from 0.050wt% sodium hydroxide to 0.50wt% sodium hydroxide, the contents of converted Polygala tenuifolia saponins and 3,4,5-trimethoxycinnamic acid increased significantly. When the alkali concentration was further increased to 0.15wt% sodium hydroxide, the content of Polygala tenuifolia saponins did not increase significantly, indicating that 0.15wt% sodium hydroxide treatment can already meet the full conversion of Polygala tenuifolia saponins.

[0205] Considering that the content of another active ingredient, 3,4,5-trimethoxycinnamic acid, decreases significantly with further increase in alkali treatment concentration, the yield of 3,4,5-trimethoxycinnamic acid is the highest when treated with a medium concentration of 0.15wt% sodium hydroxide, and the optimal alkali treatment concentration is determined to be 0.150wt% sodium hydroxide.

[0206] Under the same conditions, increasing the reaction temperature from 50°C to 115°C significantly increased the contents of polygala tenuifolia saponins and 3,4,5-trimethoxycinnamic acid, and the optimal reaction temperature was determined to be 115°C.

[0207] Under the same conditions, extending the reaction time from 1 hour to 4 hours slightly increased the content of Polygala tenuifolia saponins and 3,4,5-trimethoxycinnamic acid. However, the increase was greater at low temperatures, while the increase was minimal at high temperatures, such as 115°C. Considering production efficiency, the optimal reaction time was determined to be 1 hour.

[0208] Example 3: Optimization of Polygala tenuifolia Alcohol Extraction Process

[0209] A polygala alcohol extraction process scheme, the specific steps are as follows:

[0210] Step 1: Grind 20g of dried Polygala tubes using a grinder and sieve through a 100-mesh sieve to obtain Polygala powder. Soak 8g of the powder in 80mL of water overnight, divide the mixture into 8 equal portions, centrifuge each portion, and collect the precipitate.

[0211] Step 2: Take one portion of the above precipitate, add 5 mL of 75% vv ethanol, mix well, soak for 12 hours, and centrifuge to separate the supernatant. Take another portion of the precipitate, add 10 mL of 75% vv ethanol, mix well, soak for 12 hours, and centrifuge to separate the supernatant.

[0212] Step 3: Take one portion of the above precipitate and add 5 mL of 95% vv ethanol, mix well, soak for 12 hours, and centrifuge to separate the supernatant. Take another portion of the precipitate and add 10 mL of 95% vv ethanol, mix well, soak for 12 hours, and centrifuge to separate the supernatant.

[0213] Step 4: Take the four portions of precipitate, dry them at 60°C, and crush them. Take one portion of the dried precipitate, add 5 mL of 75% v / v ethanol, mix well, soak for 12 hours, and centrifuge to separate the supernatant. Take another portion of the precipitate, add 10 mL of 75% v / v ethanol, mix well, soak for 12 hours, and centrifuge to separate the supernatant.

[0214] Take one portion of the dried precipitate and add 5 mL of 95 vv% ethanol, mix well, soak for 12 hours, and centrifuge to separate the supernatant. Take another portion of the dried precipitate and add 10 mL of 95 vv% ethanol, mix well, soak for 12 hours, and centrifuge to separate the supernatant.

[0215] Step 5: The 8 treated samples were tested for 3,6'-diesinapoylsucrose and polygala tenuifolia ketone III content. The test results are shown in Table 4.

[0216] Table 4: Optimization of Polygala tenuifolia Alcohol Extraction Process

[0217]

[0218] As can be seen from Table 4, under the same conditions, when a lower concentration of 75 vv% ethanol was used for extraction, the active ingredients 3,6'-dieserinoylsucrose and polygala tenuifolia ketone III could not be detected in the sample. Instead, a high concentration of ethanol, i.e., 95 vv% ethanol, could extract the active ingredients 3,6'-dieserinoylsucrose and polygala tenuifolia ketone III. This may be because 3,6'-dieserinoylsucrose and polygala tenuifolia ketone III have poor solubility in water.

[0219] Under the same conditions, increasing the solvent volume from 5 mL to 10 mL slightly increased the content of polygala tenuifolia ketone III, but the content of 3,6'-diesinapoylsucrose fluctuated slightly. The optimal solvent addition ratio was determined to be 10 mL of ethanol: 1 g of raw material.

[0220] Under the same conditions, drying the aqueous extract significantly increased the content of the two active ingredients, by approximately fourfold, and achieved the highest yields of both active ingredients. This is likely due to the high water content of the precipitate from the current aqueous extraction method. Furthermore, although the water content of the system after drying and then adding 75% vv ethanol was higher than that of the system after drying and then adding 95% vv ethanol, the yields of 3,6'-diesinapoylsucrose and polygalactone III were still higher in the former. This suggests that drying the aqueous extract not only reduces the water content and increases the yield, but also facilitates the release of 3,6'-diesinapoylsucrose and polygalactone III, further improving the yield. Therefore, this process preferably involves drying the solid portion after aqueous extraction and then adding 95% vv ethanol for extraction, achieving the highest yields of 3,6'-diesinapoylsucrose and polygalactone III.

[0221] Example 4: Extraction process of co-extraction method of active substances in Polygala tenuifolia

[0222] A co-extraction scheme for active substances of Polygala tenuifolia, the specific steps are as follows:

[0223] Step 1: Take 1 kg of cored Polygala tubes, soak them in 1 kg / 10 L of water, and perform ultrasonic cleaning for 1 to 5 minutes. Oven dry at 60°C, crush them in a grinder, and pass them through a 100-mesh sieve.

[0224] Step 2: Put the above-mentioned Polygala powder into a clean container, add 10L of drinking water, stir until the powder is completely soaked, and place it at 2-8°C overnight.

[0225] Step 3: Take the mixed solution and filter it through a 5 μm filter membrane to separate the liquid and solid precipitate.

[0226] Step 3: Transfer the above liquid to a 20L reaction tank, add 0.15wt% sodium hydroxide (pH about 10), stir to dissolve, heat to 115℃ for 1h, add 0.25% citric acid to neutralize to pH=6, filter through a 100-mesh sieve, and collect the filtrate as the water extract.

[0227] Step 4: Take the solid precipitate obtained in step 2 above, dry it at 60°C, crush it, add 10L of 95% ethanol, soak it overnight, filter it through a 5μm filter membrane, and collect the clarified liquid, which is the alcohol extract.

[0228] Step 5: Combine 10 L of the aqueous extract and 10 L of the alcohol extract, make up to 50 L with purified water, add 5 wt% maltodextrin, and spray dry using a spray dryer. Spray drying conditions are: inlet air temperature 170°C, feed pressure 0.2 bar, and feed rate 30 rpm / min. Collect the resulting powder to obtain the co-extracted active ingredient powder of Polygala tenuifolia (approximately 1000 g), which is designated as sample E4.

[0229] Step 6: Dissolve 1 g of the above powder in 1 ml of methanol, filter, and test the contents of total saponins, Polygala tenuifolia saponins, 3,4,5-trimethoxycinnamic acid, 3,6'-diesinapoylsucrose, and Polygala tenuifolia ketone III.

[0230] The yield of total saponins was 3.5%, the yield of Polygala tenuifolia saponins was 0.441%, the yield of 3,4,5-trimethoxycinnamic acid was 0.051%, the yield of 3,6'-diesinapoylsucrose was 0.22%, and the yield of Polygala tenuifolia ketone III was 0.084%. Specific data are shown in the table below.

[0231] Table 5: Polygala tenuifolia powder test data

[0232]

[0233] Example 5: Efficacy test of polygala co-extracts in improving sleep in mice

[0234] The sleep-improving activity (shortening the sodium pentobarbital sleep latency and prolonging the sodium pentobarbital sleep time) of Polygala tenuifolia powder (purchased from Anhui Kangweifu Pharmaceutical Co., Ltd.), group C1 and group C2 obtained in Example 1, and group E4 obtained in Example 4 were measured.

[0235] Step 1: Dosing of mice: After adult female mice were placed in an animal room and acclimated for one week, 50 mice were randomly divided into five groups, with 10 mice in each group. The first group was a blank control group and was given normal saline (0.2 mL / mouse). The second group was given an aqueous solution of Polygala tenuifolia powder (5g / 100ml water soaked) (0.2 mL / mouse). The third group was given the group C1 sample obtained in Example 1 (5g / 100ml water dissolved) (0.2 mL / mouse). The fourth group was given the group C2 sample obtained in Example 1 (5g / 100ml water dissolved) (0.2 mL / mouse). The fifth group was given the group E4 sample obtained in Example 4 (5g / 100ml water reconstituted) (0.2 mL / mouse). Administration was by gavage once a day for 30 consecutive days.

[0236] Step 2: Each group of animals was intraperitoneally injected with sodium pentobarbital at a dose of 45 mg / kg BW. The disappearance of righting reflex was used as an indicator to record the time of starting intraperitoneal injection, sleep onset, sleep duration, and wakefulness of the mice.

[0237] Step 3: Count and analyze the sleep latency and sleep time of each group of mice. The specific results are as follows: Figure 1 、 2 and shown in Table 6.

[0238] Table 6: Results of sleep improvement experiment

[0239]

[0240] As can be seen, Group 5 (i.e., the group administered with the E4 sample) significantly outperformed the other control groups in both the number of animals that fell asleep and the duration of sleep, achieving nearly 100% sleep onset and a shorter sleep duration. Sleep duration was also significantly superior to the other control groups. This demonstrates that the co-extract of the active ingredients from Polygala tenuifolia obtained in this invention exhibits excellent effects in shortening the sodium pentobarbital sleep latency and prolonging the sodium pentobarbital sleep duration.

Claims

1. A co-extract of active ingredients from Polygala tenuifolia, characterized in that: The polygala active ingredient co-extract comprises active ingredients selected from the group consisting of polygala tenuifolia saponins, polygala tenuifolia ketone, 3,6'-diesinapoylsucrose and 3,4,5-trimethoxycinnamic acid; The content of polygala tenuifolia saponin is ≥0.10 mg / mL; the content of 3,6' dierucyl sucrose is ≥0.05 mg / mL; the content of 3,4,5-trimethoxycinnamic acid is ≥0.01 mg / mL; and the content of polygala tenuifolia ketone is ≥0.02 mg / mL. The co-extract of the active ingredients of Polygala tenuifolia is prepared by a method comprising the following steps: (1) Providing raw materials of Polygala tenuifolia, drying and crushing them to obtain Polygala tenuifolia powder; (2) Take the Polygala powder obtained in step (1), extract it with water to obtain the Polygala water extract, filter it, and collect the supernatant and solid part respectively: (3) Take the supernatant obtained in step (2), add alkaline solution to react, filter, and obtain alkaline-treated filtrate and filter cake: (4) Take the filter cake obtained in step (3), dry and crush it, add 70-98 vv% ethanol to extract it, and filter it to obtain the alcohol extraction supernatant and filter cake: (5) combining the alcohol extraction supernatant obtained in step (4) and the alkali-treated filtrate obtained in step (3) to obtain a co-extract of the active ingredients of Polygala tenuifolia; The drying temperature in step (4) is 55-65°C; The concentration of the alkaline solution in step (3) is 0.01-0.5 wt %.

2. The polygala root active ingredient co-extract according to claim 1, characterized in that The polygala tenuifolia saponin content of the polygala tenuifolia active ingredient co-extract is ≥0.14 mg / mL.

3. The polygala root active ingredient co-extract according to claim 1, wherein The polygala active ingredient co-extract further has one or more characteristics selected from the following group: (i) 3,6'-diesinapoylsucrose content ≥ 0.07 mg / mL; (ii) 3,4,5-trimethoxycinnamic acid content ≥ 0.018 mg / mL; (iii) The content of polygala tenuifolia ketone is ≥ 0.028 mg / mL.

4. The polygala root active ingredient co-extract according to claim 1, characterized in that The raw material in step (1) is cored Polygala tube.

5. The polygala root active ingredient co-extract according to claim 1, wherein The step (5) further includes: The alcohol extraction supernatant (second filtrate) obtained in step (4) and the alkali-treated filtrate (first filtrate) obtained in step (3) are mixed and diluted with water to obtain a dilution, and 2-10 wt% maltodextrin is added to the dilution, and the mixture is spray-dried to obtain a co-extract of the active ingredients of Polygala tenuifolia.

6. The polygala root active ingredient co-extract according to claim 1, characterized in that The concentration of ethanol in step (4) is 90-98 vv%.

7. The polygala root active ingredient co-extract according to claim 1, wherein The concentration of the alkaline solution in step (3) is 0.1~0.2wt%.

8. The polygala root active ingredient co-extract according to claim 1, wherein The alkaline solution in step (3) is sodium hydroxide.

9. The use of the co-extract of the active ingredients of Polygala tenuifolia according to claim 1, characterized in that: Used for preparing a composition for improving sleep.

10. A composition, characterized in that The composition comprises: (1) the co-extract of the active ingredients of Polygala tenuifolia as claimed in claim 1; and (2) additional ingredients that can be used in food or medicine.

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