Polygala tenuifolia active ingredient co-extract as well as preparation and application thereof

Abstract

The invention provides a polygala tenuifolia active ingredient co-extract and preparation and application thereof, specifically, the polygala tenuifolia active ingredient co-extract comprises tenuigenin, polygalone III, 3, 6 '-dimustard acyl sucrose and 3, 4, 5-trimethoxy cinnamic acid.According to the preparation method of the co-extract, water-soluble and alcohol-soluble active ingredients can be extracted at the same time, and meanwhile, the coextract can be used for preparing the polygala tenuifolia active ingredient co-extract. It is also ensured that tenuigenin is converted into tenuigenin, and loss of other active ingredients is avoided. The polygala tenuifolia active ingredient co-extract can obviously improve the sleep quality of mice, and the effect of the polygala tenuifolia active ingredient co-extract is superior to that of the existing polygala tenuifolia powder and other single active ingredients such as a tenuifolin extraction ingredient or a polygala tenuifolia ketone III extraction ingredient.

Description

Technical Field

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

[0002] Polygala tenuifolia Willd., scientifically known as Polygala tenuifolia Willd., belongs to the Polygalaceae family, and its root is used as medicine. Polygala grows on sunny hillsides or roadsides, and is mainly distributed in Northeast China, North China, Northwest China, Shandong, Anhui, Jiangxi, Jiangsu and other places. Polygala tube is cylindrical, hollow, cramped and not straight, 3 to 12 cm long, 0.3 to 1 cm in diameter. The surface is gray or grayish yellow, with dense and deep horizontal wrinkles all over. The cross section is yellowish-white, relatively flat, with a slight smell of green grass, bitter and slightly spicy, and has a piercing throat. Polygala has the effects of calming the mind, improving intelligence, removing phlegm, and reducing swelling. It is used for insomnia, dreaminess, forgetfulness, palpitations, confusion, coughing and expectoration, sores, swelling and poison, and breast swelling and pain caused by disharmony between the heart and kidney.

[0003] The characteristic active ingredients of Polygala extract mainly include triterpenoid saponins, ketones, oligosaccharides and other active ingredients. Polygala saponin is one of the main active ingredients of Polygala, which is a pentacyclic triterpenoid saponin of oleanolic acid type, with anti-inflammatory, anti-oxidative and anti-aging, neuroprotective and anti-depressant effects. The structural characteristics of these saponin compounds are: C-2 is often substituted by hydroxyl, C-12 and 13 are unsaturated double bonds; and most of them are disaccharide chains, C-3 is connected to glucose, and C-28 is connected to sucrose through an ester bond. This type of saponin generally has a hemolytic effect, and after the C-28 ester bond is hydrolyzed by alkaline hydrolysis, the hemolytic effect disappears and the toxicity is greatly reduced, that is, the conversion of Polygala saponin into Polygala tenuifolia saponin can eliminate the toxic effect of saponin. In addition, Polygala tenuifolia saponin has strong chemical stability and strong anticholinesterase activity, and is significantly superior to Polygala saponin in improving the efficacy of neurological function. Xanthone, also known as benzochromone, has diuretic, antibacterial, anticancer, and antidepressant activities. More than 80 xanthone compounds have been isolated from Polygala plants, mainly in the form of simple xanthone compounds and xanthone glycosides. The oligosaccharide ester components contained in Polygala plants mainly use sucrose as the common mother nucleus, and on this basis, glucose (a few are rhamnose) is connected with different forms of glycosidic bonds, such as dierucyl sucrose, which has antidepressant, neuroprotective, neuronal regeneration, and memory improvement effects. Other components, such as 3,4,5-trimethoxycinnamic acid and methyl 3,4,5-trimethoxycinnamate, have sedative and hypnotic activities. Polygala saponins and other structures contain the structure of 3,4,5-trimethoxycinnamic acid, which is one of the products of Polygala saponins converted from Polygala tenuifolia saponins.

[0004] However, when taking Polygala tenuifolia Willd. using the conventional decocting method, the active substances of Polygala tenuifolia Willd. cannot be fully obtained, and only a small part of the active components taken is from Polygala tenuifolia Willd. Taking Polygala tenuifolia Willd. powder directly is also a common method. However, the absorption efficiency of direct Polygala tenuifolia Willd. powder is low, and a large amount of polygala saponins have not been converted into tenuifoliside I, which has relatively high toxicity and poor curative effect. Therefore, there have been reports on extracting tenuifoliside I using the alkali hydrolysis method, or extracting polygalaxanthone III and oligosaccharide lipids using alcohol extraction. However, neither the alkali hydrolysis method nor the alcohol extraction method can obtain tenuifoliside I, polygalaxanthone III, and oligosaccharide lipids simultaneously. High-concentration alkali treatment easily leads to the instability of substances such as polygalaxanthone III, oligosaccharide lipids, and the conversion product of polygala saponins, 3,4,5-trimethoxycinnamic acid. Therefore, in the currently published materials, there are few reports on the method for co-extracting active substances such as tenuifoliside I, polygalaxanthone III, 3,6'-di-O-sinapoyl-sucrose, and 3,4,5-trimethoxycinnamic acid. Summary of the Invention

[0005] The present invention provides a co-extract of active components of Polygala tenuifolia Willd. including a variety of active components such as tenuifoliside I, polygalaxanthone III, 3,6'-di-O-sinapoyl-sucrose, and 3,4,5-trimethoxycinnamic acid, and its preparation and application. The preparation method ensures a high conversion rate of tenuifoliside I while ensuring the stability of polygalaxanthone III, 3,6'-di-O-sinapoyl-sucrose, and 3,4,5-trimethoxycinnamic acid.

[0006] In the first aspect of the present invention, there is provided a co-extract of active components of Polygala tenuifolia Willd., which co-extract of active components of Polygala tenuifolia Willd. includes active components selected from the group consisting of: tenuifoliside I, polygalaxanthone III, 3,6'-di-O-sinapoyl-sucrose, and 3,4,5-trimethoxycinnamic acid.

[0007] In another preferred example, the content of tenuifoliside I in the co-extract of active components of Polygala tenuifolia Willd. is ≥ 0.10 mg / mL.

[0008] In another preferred example, the content of tenuifoliside I in the co-extract of active components of Polygala tenuifolia Willd. is ≥ 0.14 mg / mL; preferably, ≥ 0.16 mg / mL.

[0009] In another preferred example, the co-extract of active components of Polygala tenuifolia Willd. further has one or more characteristics selected from the group consisting of: (i) The content of 3,6'-di-O-sinapoyl-sucrose is ≥ 0.05 mg / mL; (ii) The content of 3,4,5-trimethoxycinnamic acid is ≥ 0.01 mg / mL; (ii) The content of polygalaxanthone III is ≥ 0.02 mg / mL.

[0010] In another preferred embodiment, the content of 3,6'-di-sinapoyl sucrose in the co-extract of polygala tenuifolia active ingredients is ≥ 0.07 mg / mL; preferably, ≥ 0.08 mg / mL.

[0011] In another preferred embodiment, the content of 3,4,5-trimethoxycinnamic acid in the co-extract of polygala tenuifolia active ingredients is ≥ 0.018 mg / mL; preferably, ≥ 0.020 mg / mL.

[0012] In another preferred embodiment, the content of tenuifolin III in the co-extract of polygala tenuifolia active ingredients is ≥ 0.028 mg / mL; preferably, ≥ 0.03 mg / mL.

[0013] In another preferred embodiment, the content of total saponins in the co-extract of polygala tenuifolia active ingredients is ≥ 1 mg / mL; preferably, ≥ 1.2 mg / mL.

[0014] In another preferred embodiment, the co-extract of polygala tenuifolia active ingredients is prepared by the following method: (1) Provide polygala tenuifolia raw materials, dry and crush them to obtain polygala tenuifolia powder; (2) Take the polygala tenuifolia powder obtained in step (1), extract it with water to obtain a polygala tenuifolia water extract, filter it, and separately collect the supernatant and the solid part: (3) Take the supernatant obtained in step (2), add an alkali solution to react, filter it, and obtain an alkali-treated filtrate (the first filtrate) and a filter cake: (4) Take the filter cake obtained in step (3), dry and crush it, add 70-98 vv% ethanol for extraction, filter it, and obtain an alcohol extraction supernatant (the second filtrate) and a filter cake: (5) Combine the alcohol extraction supernatant (the second filtrate) obtained in step (4) and the alkali-treated filtrate (the first filtrate) obtained in step (3) to obtain the co-extract of polygala tenuifolia active ingredients.

[0015] In the second aspect of the present invention, there is provided a method for preparing the co-extract of polygala tenuifolia active ingredients as described in the first aspect of the present invention, and the method includes: (1) Provide polygala tenuifolia raw materials, dry and crush them to obtain polygala tenuifolia powder; (2) Take the polygala tenuifolia powder obtained in step (1), extract it with water to obtain a polygala tenuifolia water extract, filter it, and separately collect the supernatant and the solid part: (3) Take the supernatant obtained in step (2), add an alkali solution to react, filter it, and obtain an alkali-treated filtrate (the first filtrate) and a filter cake: (4) Take the filter cake obtained in step (3), dry and crush it, add 70-98 vv% ethanol for extraction, filter it, and obtain an alcohol extraction supernatant (the second filtrate) and a filter cake: (5) Combine the alcohol extraction supernatant (the second filtrate) obtained in step (4) and the alkali treatment filtrate (the first filtrate) obtained in step (3) to obtain the co-extract of the active ingredients of Polygala tenuifolia Willd..

[0016] In another preferred embodiment, step (5) further includes: After the alcohol extraction supernatant (the second filtrate) obtained in step (4) and the alkali treatment filtrate (the first filtrate) obtained in step (3) are mixed, water is added for dilution to obtain a dilution solution, and 2-10 wt% of maltodextrin is added to the dilution solution, and then spray drying is performed to obtain the co-extract of the active ingredients of Polygala tenuifolia Willd..

[0017] In another preferred embodiment, the conditions for spray drying are: the inlet air temperature is between 135 and 170 °C, the feed pressure is between 0.2 and 0.4 bar, and the feed rate is between 30 and 50 rpm / min.

[0018] In another preferred embodiment, after adding water for dilution, the ethanol concentration in the dilution solution ≤ 25 wt%, preferably, the ethanol concentration in the dilution solution ≤ 20 wt%.

[0019] In another preferred embodiment, the drying temperature in step (4) is 55-65 °C.

[0020] In another preferred embodiment, the concentration of ethanol in step (4) is 90-98 v / v%.

[0021] In another preferred embodiment, the ethanol addition ratio in step (4) is that the volume of ethanol is (4-18) mL: 1 g of raw material; preferably (8-12) mL: 1 g of raw material.

[0022] In another preferred embodiment, the ethanol extraction temperature in step (4) is 0-28 °C; preferably, it is 15-25 °C.

[0023] In another preferred embodiment, the concentration of the alkali solution in step (3) is 0.01-0.5 wt%.

[0024] In another preferred embodiment, the concentration of the alkali solution in step (3) is 0.1-0.2 wt%.

[0025] In another preferred embodiment, the alkali solution in step (3) is sodium hydroxide.

[0026] In another preferred embodiment, the pH of the solution after adding the alkali solution in step (3) is 8-12.

[0027] In another preferred embodiment, the reaction temperature in step (3) is 100-150 °C; preferably 110-120 °C.

[0028] In another preferred embodiment, the reaction time in step (3) is 0.2-5 h; preferably 0.5-1.5 h.

[0029] In another preferred example, step (3) further includes: adding citric acid to adjust the pH of the solution back to about 5-8, preferably pH = 6.

[0030] In another preferred example, the raw material in step (1) is defatted polygala root.

[0031] In another preferred example, step (1) further includes ultrasonic cleaning of the raw material: In another preferred example, step (1) further includes: soaking in water at 1 kg / (8-12) L and performing ultrasonic cleaning for 1-10 minutes.

[0032] In another preferred example, the drying temperature in step (1) is 55-65 °C.

[0033] In another preferred example, the water addition ratio in step (1) is (8-12) L: 1 kg of raw material.

[0034] In another preferred example, the water extraction temperature in step (1) is 0-28 °C, preferably 2-15 °C.

[0035] In another preferred example, the water extraction time in step (1) is 2-24 hours, preferably 10-15 hours.

[0036] In the third aspect of the present invention, there is provided the use of the co-extract of polygala active ingredients as described in the first aspect of the present invention for preparing a composition for improving sleep.

[0037] In another preferred example, the composition has one or more of the following characteristics selected from the group: (a) shortening the pentobarbital latent sleep period; (b) prolonging the pentobarbital sleep time.

[0038] In another preferred example, the co-extract of polygala active ingredients has the effect of improving sleep.

[0039] In another preferred example, the co-extract of polygala active ingredients has the effect of shortening the pentobarbital latent sleep period and / or prolonging the pentobarbital sleep time.

[0040] In the fourth aspect of the present invention, there is provided a composition, which includes: (1) the co-extract of polygala active ingredients as described in the first aspect of the present invention; and (2) additional ingredients that can be used in food or medicine.

[0041] It should be understood that within the scope of the present invention, the above technical features of the present invention and the technical features specifically described below (such as in the examples) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be elaborated one by one here. Description of the Drawings

[0042] Figure 1 The experimental results of various Polygala tenuifolia extracts shortening the sleep latency of sodium pentobarbital Figure 1 The above figure shows the number of sleeping animals Figure 1 The following figure shows the sleep time

[0043] Figure 2 The experimental results of various Polygala tenuifolia extracts prolonging the sleep time of sodium pentobarbital Detailed implementation mode

[0044] After extensive and in-depth research, the inventor of the present invention unexpectedly developed for the first time a co-extract of Polygala tenuifolia active components containing multiple Polygala tenuifolia active components and having a remarkable sleep-improving effect. The co-extract of Polygala tenuifolia active components contains four key Polygala tenuifolia active components: tenuifoliside A, polygalaxanthone III, 3,6'-di-O-sinapoyl-sucrose, and 3,4,5-trimethoxycinnamic acid. The preparation method of the co-extract of Polygala tenuifolia active components includes water extraction and ethanol extraction, ensuring that both water-soluble active substances (tenuifoliside) and water-insoluble substances (polygalaxanthone III, 3,6'-di-O-sinapoyl-sucrose) in Polygala tenuifolia can be extracted, and a low-concentration base is used in the preparation method, which is safe and low-pollution. On this basis, the present invention is completed.

[0045] The main advantages of the present invention: 1. Superior activity: The co-extract of Polygala tenuifolia active components described in the present invention includes four key Polygala tenuifolia active components: tenuifoliside A, polygalaxanthone III, 3,6'-di-O-sinapoyl-sucrose, and 3,4,5-trimethoxycinnamic acid, and it is confirmed in animal experiments that the effect of improving the sleep quality of mice is the best.

[0046] 2. High content of active components and less degradation: The method described in the present invention includes water extraction and ethanol extraction, ensuring that both water-soluble active substances (tenuifoliside) and water-insoluble substances (polygalaxanthone III, 3,6'-di-O-sinapoyl-sucrose) in Polygala tenuifolia can be extracted. Different from the reported extraction method of tenuifoliside (directly adding ethanol after alkali hydrolysis), in this process, the water extract and the solid residue are separated before alkali treatment, and the solid residue is extracted with ethanol. Tests show that this method can avoid the hydrolysis of active substances such as polygalaxanthone III and 3,6'-di-O-sinapoyl-sucrose under alkaline conditions.

[0047] 3. The preparation method is safe and effective: The method described in the present invention has a low alkali treatment concentration (preferably 0.15wt% sodium hydroxide), a pH of about 10, and a treatment temperature of 100-115°C, which is different from the prior art that requires a high concentration of alkali. The method has low requirements for industrial facilities and low potential risks to operators. At the same time, it can also ensure a higher conversion rate of Polygala tenuifolia saponins and the highest stability of other active ingredients such as 3,4,5-trimethoxycinnamic acid.

[0048] 4. Unexpected effect of drying: The present invention also unexpectedly found that after the solid part of the water extraction of Polygala was dried at 60°C for more than 3 hours, and then extracted with ethanol water of not less than 75%, the extraction yield of Polygala tenuifolia ketone III and 3,6'-dierucyl sucrose was significantly improved. And through comparative experiments, it was found that the process of drying the water extract can not only reduce the moisture content and increase the extraction yield of active ingredients, but also help release 3,6'-dierucyl sucrose and Polygala tenuifolia ketone III, further improving the yield. At the same time, the ethanol soaking process can save the complicated process of heating and reflux, and the method is simple.

[0049] the term In order to more easily understand the present invention, some 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 commonly 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 be changed. 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.

[0050] 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.

[0051] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are only intended to illustrate the present invention and are not intended to limit the scope of the present invention. Unless otherwise stated, percentages and parts are weight percentages and weight fractions.

[0052] General detection method

[0053] Total saponins detection method

[0054] (1) Principle The total saponins of 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 substance.

[0055] (2) Reagents and equipment Developer: 0.5% vanillin glacial acetic acid solution (0.5 g → 100 mL glacial acetic acid) Reaction acid: Perchloric acid (analytical grade) Standard: Ginsenoside Re (purity ≥ 98%) (C 48 H 82 O 18 , CAS No.: 257 - 814 - 6) Solvents: Methanol, absolute ethanol, glacial acetic acid Equipment: UV - visible spectrophotometer, constant temperature water bath (3) Solution preparation Preparation of standard stock solution: Accurately weigh 10 mg of ginsenoside Re standard, accurate to 0.00001 g, and dissolve it in methanol to prepare a 1 mg / mL standard stock solution, and store it in a refrigerator at 4°C for 6 months.

[0056] Vanillin solution: Take 0.5 g of vanillin (C8H8O3), dissolve it in glacial acetic acid and make up the volume to 100 mL.

[0057] (4) Analysis Standard working solution: Take 0 µL, 20 µL, 40 µL, 60 µL, 80 µL, 100 µL of the standard stock solution (equivalent to 0 µg, 20 µg, 40 µg, 60 µg, 80 µg, 100 µg) and place them in 10 mL colorimetric tubes respectively. Evaporate to dryness in a water bath at 60°C and set aside.

[0058] Preparation of test sample solution: Take 10 - 50 µl of the test sample solution, place it in a centrifuge tube, heat it to dryness in a constant temperature water bath at 60°C and set aside.

[0059] Drawing of 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 constant temperature water bath at 60°C for 10 min, take out, cool in an ice bath, add glacial acetic acid to make up the volume to 5.0 mL, shake well, and perform colorimetry at a wavelength of 560 nm with a 1 cm colorimetric cell to obtain the absorbance value. Plot the standard curve with the concentration as the abscissa and the absorbance value as the ordinate.

[0060] (5) Determination In the test tube, refer to the "Drawing of standard working curve" in step (4) to obtain the absorbance value, and calculate the content of yuanzhi saponin in the test sample solution from the standard curve.

[0061] Blank test: Except for not adding the test sample, perform the determination according to the determination operation steps.

[0062] (6) Result calculation:

[0063] In the formula: X — The content of total polygala root saponins in the sample, in grams per 100 grams (g / 100 g or g / 100 mL); A — The mass of total polygala root saponins in the test solution calculated from the standard curve, in micrograms (µg); V1 — The total volume of the sample preparation, in milliliters (mL); V2 — The volume of the sample preparation solution for determination, in milliliters (mL); m — The mass or volume of the sample, in grams or milliliters (g or mL).

[0064] The calculation result is retained to two significant figures.

[0065] Detection method for tenuifolin

[0066] (1) Reference documents: It is determined in accordance with the "Content Determination" of "Medicinal Materials and Slices - Polygala Root" in Part I of the Chinese Pharmacopoeia and the "General Rules 0512 High Performance Liquid Chromatography" in Part IV of the Chinese Pharmacopoeia.

[0067] (2) Reagents and equipment Standard product: Tenuifolin standard product (C 36 H 56 O 12 , CAS No.: 20183 - 47 - 5, purity ≥ 98%) Reagents: Methanol, phosphoric acid (chromatographically pure); Wahaha purified water Equipment: Shimadzu high performance liquid chromatograph LC - 2030, ECLIPSE PLUS C18 (3.5μm, 4.6×100mm) (3) Solution preparation Preparation of 1mg / mL standard stock solution: Accurately weigh 10mg of tenuifolin standard product, accurate to 0.00001g, and prepare a 1mg / mL standard stock solution with methanol. Store it in a refrigerator at 4°C for 6 months.

[0068] Preparation of a series of reference substance solutions: Take 0µL, 50μL, 100µL, 200µL, 400µL, 800µL of the standard stock solution and place them in EP tubes respectively, add methanol to 1mL, to obtain tenuifolin solutions of 0.0mg / mL, 0.05mg / mL, 0.1mg / mL, 0.2mg / mL, 0.4mg / mL and 0.8mg / mL. All concentration reference substance solutions are filtered through a 0.45μm filter membrane to obtain.

[0069] Preparation of the test solution: Take 1mL of the test sample and filter it through a 0.45μm filter membrane to obtain.

[0070] (4)Chromatographic conditions 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.

[0071] (5) Analysis Accurately measure 10 μL of the reference solution and the test solution respectively, inject them into the liquid chromatograph, and record the chromatogram.

[0072] (6) Data processing The raw data generated by the high performance liquid chromatograph are automatically integrated and peak marked by the instrument's own software.

[0073] (7) Result processing and calculation Linear regression was performed on the peak area measured with the reference solution of Polygala tenuifolia saponins of various concentrations (mg / mL) to obtain the linear regression equation Y=bx+a. The linear regression correlation coefficient r should not be less than 0.99.

[0074] Substitute the measured peak area of the test sample into the linear regression equation to calculate the content of Polygala tenuifolia saponin in the test sample (mg / mL).

[0075] 3,6'--Dierucyl Sucrose Detection Method

[0076] (1) Reference documents: Determined in accordance with the "Content Determination" of "Chinese Pharmacopoeia Volume 1" "Medicinal Materials and Decoction Pieces Polygala" and "General Chapter 0512 High Performance Liquid Chromatography" of "Chinese Pharmacopoeia Volume 4".

[0077] (2) Reagents and equipment Standard 3,6'-Dierucyl Sucrose Standard (C 34 H 42 O 19 , CAS No.: 139891-98-8, purity ≥98%) Reagents: acetonitrile, phosphoric acid (chromatographic grade); Wahaha purified water Equipment: Shimadzu HPLC LC-2030, ECLIPSE PLUS C18 (3.5μm, 4.6×100mm) (3) Solution preparation Preparation of 1 mg / mL standard stock solution: Accurately weigh 10 mg of 3,6'-dicerinoylsucrose standard to an accuracy of 0.00001 g, prepare it into 1 mg / mL standard stock solution with methanol, and store it in a 4°C refrigerator for 6 months.

[0078] Preparation of series of reference solutions: 0µL, 50µL, 100µL, 200µL, 400µL, and 800µL of the standard stock solution were placed in EP tubes respectively, and methanol was added to 1ml, that is, 0mg / mL, 0.050mg / mL, 0.100mg / mL, 0.200mg / mL, 0.400mg / mL, and 0.800mg / mL 3,6'-dicerinoylsucrose solutions. All concentration reference solutions were filtered through a 0.45μm filter membrane.

[0079] Preparation of test solution: Take 1 mL of the test sample and filter it through a 0.45 μm filter membrane.

[0080] (4) Chromatographic conditions 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.

[0081] (5) Analysis Accurately measure 10 μl of the reference solution and the test solution respectively, inject them into the liquid chromatograph, and record the chromatogram.

[0082] (6) Data processing The raw data generated by the high performance liquid chromatograph are automatically integrated and peak marked by the instrument's own software.

[0083] (7) Result processing and calculation Perform linear regression on the measured peak area using the 3,6'-dicerucyl sucrose reference solution (mg / mL) at various concentrations to obtain the linear regression equation Y=bx+a. The linear regression correlation coefficient r should not be less than 0.99.

[0084] Substitute the measured peak area of the test sample into the linear regression equation to calculate the 3,6'-dicerinoylsucrose content (mg / mL) in the test sample.

[0085] 3,4,5-Trimethoxycinnamic acid content detection method

[0086] (1) References: Zhao Mengjun, Li Chenchen, Gan Long, et al. Determination of the contents of eight components of Polygala tenuifolia by HPLC[J]. Modern Chinese Medicine, 2020, 22(3)

[0087] Standard 3,4,5-Trimethoxycinnamic acid standard (C 12 H 14 O5, CAS No.: 90-50-6, purity ≥ 98%) Reagents: acetonitrile, phosphoric acid (chromatographic grade); Wahaha purified water Equipment: Shimadzu HPLC LC-2030, ECLIPSE PLUS C18 (3.5μm, 4.6×100mm) (3) Solution preparation: Preparation of 1 mg / mL standard stock solution: Accurately weigh 10 mg of 3,4,5-trimethoxycinnamic acid standard to an accuracy of 0.00001 g, prepare it into 1 mg / mL standard stock solution with methanol, and store it in a 4°C refrigerator for 6 months.

[0088] Preparation of series of reference solutions: 0µL, 20µL, 40µL, 80µL, 160µL and 320µL of standard stock solution were placed in EP tubes respectively, and methanol was added to 1mL to obtain 0.0mg / mL, 20μg / mL, 40μg / mL, 80μg / mL, 160μg / mL and 320μg / mL 3,4,5-trimethoxycinnamic acid solutions. All concentration reference solutions were filtered through a 0.45μm filter membrane.

[0089] Preparation of test solution: Take 1 ml of the test sample and filter it through a 0.45 μm filter membrane.

[0090] (4) Chromatographic conditions The mobile phase was acetonitrile (A)-0.05% phosphoric acid-water solution (B), gradient elution (0~5 min, 25%A; 6~25min, 25%~45%A; 26~30min, 45%~80%A; 31~35min, 80%A); the flow rate was 0.5 mL / min; the column temperature was 30℃; the detection wavelength was 310 nm; the chromatographic column was ECLIPSE PLUS C18 (3.5μm, 4.6×100mm); the column temperature was 30℃; the acquisition time was 30 minutes.

[0091] (5) Analysis Accurately measure 10 μL of the reference solution and the test solution respectively, inject them into the liquid chromatograph, and record the chromatogram.

[0092] (6) Data processing The raw data generated by the high performance liquid chromatograph are automatically integrated and peak marked by the instrument's own software.

[0093] (7) Result processing and calculation Perform linear regression on the measured peak area using the reference solution of 3,4,5-trimethoxycinnamic acid at various concentrations (μg / mL) to obtain the linear regression equation Y=bx+a. The linear regression correlation coefficient r should not be less than 0.99.

[0094] Substitute the measured peak area of the test sample into the linear regression equation to calculate the content (μg / mL) of 3,4,5-trimethoxycinnamic acid in the test sample.

[0095] Determination method for polygalaxanthone Ⅲ content

[0096] (1) Reference documents: Determine according to the "Content Determination" of "Medicinal Materials and Slices - Polygala Root" in Part I of the Chinese Pharmacopoeia and the "General Rules 0512 High Performance Liquid Chromatography" in Part IV of the Chinese Pharmacopoeia.

[0097] (2) Reagents and equipment Standard Polygalaxanthone Ⅲ standard (C 25 H 28 O 15 , CAS No.: 162857-78-5, purity ≥ 98%) Reagents Methanol, phosphoric acid (chromatographic grade); Wahaha purified water Equipment Shimadzu high performance liquid chromatograph LC-2030, ECLIPSE PLUS C18 (3.5μm, 4.6×100mm) (3) Solution preparation: Preparation of 1mg / mL standard stock solution: Accurately weigh 10mg of Polygalaxanthone Ⅲ standard, accurate to 0.00001g, and dissolve it in methanol to prepare a 1mg / mL standard stock solution, and store it in a refrigerator at 4℃ for 6 months.

[0098] Preparation of series of reference solutions: Take 0µL, 20μL, 40µL, 80µL, 160µL, 320µL of the standard stock solution and place them in EP tubes respectively, add methanol to 1ml to obtain Polygalaxanthone Ⅲ solutions of 0μg / mL, 20μg / mL, 40μg / mL, 80μg / mL, 160μg / mL and 320μg / mL. Filter all concentration reference solutions with a 0.45μm filter membrane to obtain.

[0099] Preparation of test sample solution: Take 1mL of the test sample and filter it with a 0.45μm filter membrane to obtain.

[0100] (4) Chromatographic conditions Use acetonitrile - 0.05% phosphoric acid solution (18 : 82) as the mobile phase; flow rate 1.0mL / min; detection wavelength 320nm; chromatographic column: ECLIPSE PLUS C18 (3.5μm, 4.6×100mm); column temperature 30℃; collection time 35 minutes.

[0101] (5) Analysis Accurately measure 10μl of each of the reference solution and the test sample solution respectively, inject them into the liquid chromatograph, and record the chromatogram.

[0102] (6) Data processing The raw data generated by the high performance liquid chromatograph are automatically integrated and peak marked by the instrument's own software.

[0103] (7) Result processing and calculation Linear regression was performed on the peak area measured with various concentrations of polygala tenuifolia ketone III reference solution (μg / mL) to obtain the linear regression equation Y=bx+a. The linear regression correlation coefficient r should not be less than 0.99.

[0104] 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).

[0105] Example 1: Preparation of co-extracts of active ingredients from Polygala tenuifolia A selection scheme for the extraction process of Polygala tenuifolia, the specific steps are as follows: Step 1: Take 20g of dried Polygala tube and crush it with a grinder, sieve it with a 100-mesh screen to obtain Polygala powder. Divide the powder into three groups, 5g each.

[0106] Step 2: Take 5g of the polygala powder obtained in step 1 above, add 50mL of water and soak overnight, centrifuge to separate the supernatant and solid precipitate. Add 1% sodium hydroxide to the supernatant, boil at 100℃ for 1h, filter through a 100-mesh sieve to obtain a water extract. Add 50ml of anhydrous ethanol to the solid precipitate, soak overnight, and filter with filter paper to obtain an alcohol extract.

[0107] Step 3: Mix the water extract and the alcohol extract, add 5% maltodextrin, and spray dry using a spray dryer. The spray drying conditions are: inlet air temperature 140°C, feed pressure 0.2 bar, and feed speed 30 rpm / min. Collect the prepared powder, about 5 g, to obtain the group E1 sample.

[0108] 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'-dicorinoylsucrose respectively. The specific test results are shown in Table 1.

[0109] Comparative Example C1: Commonly used method for preparing and extracting Polygala tenuifolia saponins Take 5 g of the Polygala powder obtained in step 1 of Example 1, add 50 mL of water and soak overnight, collect the supernatant by centrifugation, add 10% sodium hydroxide to the supernatant, perform alkaline hydrolysis for 2 h, and adjust the pH value to 4.0 with concentrated hydrochloric acid. Concentrate the solution to 5 mL, add 45 mL of anhydrous ethanol, filter with filter paper, and obtain a filtrate.

[0110] Dilute the filtrate two times with water, add 5% maltodextrin, and spray-dry using a spray dryer. The spray-drying conditions are as follows: inlet air temperature 140 °C, feed pressure 0.2 bar, and feed rate 30 rpm / min. Collect approximately 5 g of the prepared powder. Thus, sample C1 of the group is obtained.

[0111] Take 0.1 g of the powder (1 / 50 of the total amount), dissolve it in 1 mL of methanol, and detect the contents of total saponins, tenuifoliside, and 3,6'-di-O-sinapoyl-sucrose respectively. The specific detection results are shown in Table 1.

[0112] Preparation and extraction method of the organic phase reflux extraction of Comparative Example C2 Take 5 g of the polygala tenuifolia Willd. powder obtained in Step 1 of Example 1, extract the polygala tenuifolia Willd. by the method of organic phase reflux extraction, add 50 ml of 90% ethanol, put it into an extraction container, heat it to solvent reflux, and maintain the reflux state for 1.5 hours. Conduct three times of reflux extraction. After the extraction solutions after reflux extraction are combined and cooled, make up the lost weight with 90% ethanol, shake well, filter through filter paper, and obtain the filtrate.

[0113] Dilute the filtrate two times with water, add 5% maltodextrin, and spray-dry using a spray dryer. The spray-drying conditions are as follows: inlet air temperature 140 °C, feed pressure 0.2 bar, and feed rate 30 rpm / min. Collect approximately 5 g of the prepared powder. Thus, sample C2 of the group is obtained.

[0114] Take 0.1 g of the powder (1 / 50 of the total amount), dissolve it in 1 mL of methanol, and detect the contents of total saponins, tenuifoliside, and 3,6'-di-O-sinapoyl-sucrose respectively. The specific detection results are shown in Table 1.

[0115] Table 1: Detection of the contents of substances extracted by different polygala tenuifolia Willd. extract processes

[0116] Conclusion: In Comparative Example C1, which is the preparation and extraction method of tenuifoliside, it can be seen that the content of tenuifoliside in the extract is high. This may be due to the very high concentration of sodium hydroxide, resulting in a large amount of tenuifoliside being converted. However, 3,6'-di-O-sinapoyl-sucrose was not detected, indicating that it is unable to effectively extract active components such as 3,6'-di-O-sinapoyl-sucrose with sedative and sleep-aiding effects. In Comparative Example C2, which is the reflux extraction in the organic phase, it can be observed that the total saponin content in the extract is the highest, but tenuifoliside was not detected, proving that most of them are ordinary polygala saponins and hardly contain tenuifoliside. In Example 1, which is the mild polygala transformation and extraction method described in this application, both active components, namely tenuifoliside and 3,6'-di-O-sinapoyl-sucrose, were detected in the extract, and their contents are relatively high. The two active components can be co-extracted. When converting polygala saponin into tenuifoliside, active components with sedative and sleep-aiding effects such as 3,6'-di-O-sinapoyl-sucrose can be simultaneously extracted.

[0117] Example 2: Optimization of the water extraction and alkali treatment process of Polygala tenuifolia An optimization scheme for the water extraction and alkali treatment process of Polygala tenuifolia is as follows: Step 1: Take 20 g of dried Polygala tenuifolia roots, crush them using a pulverizer, and sieve them through a 100-mesh sieve to obtain Polygala tenuifolia powder. Add the powder to 200 mL of water and soak overnight, then centrifuge and collect the supernatant.

[0118] Step 2: Take 50 mL of the above supernatant, add 0.05 wt% sodium hydroxide, dissolve it, divide it into 6 small portions, perform corresponding treatments on the 6 small portions of samples according to the conditions in Table 2, and add citric acid to adjust the pH value back to 6.0.

[0119] Table 2: Reaction conditions for sample treatment

[0120] Step 3: Take 50 mL of the above supernatant, add 0.15 wt% sodium hydroxide, dissolve it, divide it into 6 small portions, perform corresponding treatments on the 6 small portions of samples according to the conditions in Table 2, and add citric acid to adjust the pH value back to 6.0.

[0121] Step 4: Take 50 mL of the above supernatant, add 0.5 wt% sodium hydroxide, dissolve it, divide it into 6 small portions, perform corresponding treatments on the 6 small portions of samples according to the conditions in Table 2, and add citric acid to adjust the pH value back to 6.0.

[0122] Step 5: Detect the contents of tenuifoliside and 3,4,5-trimethoxycinnamic acid in the above 18 treated samples respectively. The detection results are shown in Table 3.

[0123] Table 3: Data for optimizing the alkali treatment process

[0124] As shown in Table 3 of the experimental results, it can be seen that: Under the same conditions, when the alkali treatment concentration was increased from 0.050 wt% sodium hydroxide to 0.50 wt% sodium hydroxide, the contents of tenuifolisaponin and 3,4,5-trimethoxycinnamic acid obtained by conversion both increased significantly. When the alkali concentration was further increased to 0.15 wt% sodium hydroxide, the increase in the content of tenuifolisaponin was not obvious, indicating that the treatment with 0.15 wt% sodium hydroxide could already meet the full conversion of tenuifolisaponin.

[0125] Considering that the content of another active ingredient, 3,4,5-trimethoxycinnamic acid, decreased significantly with the further increase of the alkali treatment concentration, the yield of 3,4,5-trimethoxycinnamic acid was the highest when treated with 0.15 wt% sodium hydroxide at a medium concentration. Therefore, the optimal alkali treatment concentration was determined to be 0.150 wt% sodium hydroxide.

[0126] Under the same conditions, when the reaction temperature was increased from 50 °C to 115 °C, the contents of tenuifolisaponin and 3,4,5-trimethoxycinnamic acid were both significantly increased, and the optimal reaction temperature was determined to be 115 °C.

[0127] Under the same conditions, when the reaction duration was extended from 1 h to 4 h, the contents of tenuifolisaponin and 3,4,5-trimethoxycinnamic acid were both slightly increased, but under low-temperature reaction conditions, the increase amplitude was larger, and under high temperature such as 115 °C, the increase amplitude was very small. Considering the production efficiency, the optimal reaction duration was determined to be 1 h.

[0128] Example 3: Optimization of the extraction process of polygalitol An extraction process scheme of polygalitol is as follows: Step 1: Take 20 g of dry polygala root slices, crush them with a pulverizer, and sieve them through a 100-mesh sieve to obtain polygala powder. Take 8 g of the powder, add 80 mL of water, soak it overnight, divide it into 8 portions, centrifuge them separately, and collect the precipitates.

[0129] Step 2: Take 1 portion of the above precipitate, add 5 mL of 75 v / v% ethanol, mix well and soak for 12 h, and centrifuge to separate the supernatant. Take another 1 portion of the precipitate, add 10 mL of 75 v / v% ethanol, mix well, soak for 12 h, and centrifuge to separate the supernatant.

[0130] Step 3: Take 1 portion of the above precipitate, add 5 mL of 95 v / v% ethanol, mix well and soak for 12 h, and centrifuge to separate the supernatant. Take another 1 portion of the precipitate, add 10 mL of 95 v / v% ethanol, mix well, soak for 12 h, and centrifuge to separate the supernatant.

[0131] Step 4: Take the above 4 portions of precipitates, dry them at 60 °C and crush them. Take 1 portion of the dried precipitate, add 5 mL of 75 vv% ethanol, mix well and soak for 12 h, then centrifuge to separate the supernatant. Take another 1 portion of the precipitate, add 10 mL of 75 vv% ethanol to each, mix well and soak for 12 h, then centrifuge to separate the supernatant.

[0132] Take 1 portion of the dried precipitate, add 5 mL of 95 vv% ethanol, mix well and soak for 12 h, then centrifuge to separate the supernatant. Take another 1 portion of the dried precipitate, add 10 mL of 95 vv% ethanol to each, mix well and soak for 12 h, then centrifuge to separate the supernatant.

[0133] Step 5: Detect the contents of 3,6'-di-O-sinapoyl-sucrose and tenuifolin in the above 8 processed samples respectively. The detection results are shown in Table 4.

[0134] Table 4: Optimization of the extraction process of polygala root alcohol

[0136] As can be seen from Table 4, under the same conditions, when extracting with ethanol at a lower concentration of 75 vv%, the active ingredients 3,6'-di-O-sinapoyl-sucrose and tenuifolin could not be detected in the samples. Instead, it is the ethanol at a higher concentration, i.e., 95 vv% ethanol, that can extract the active ingredients 3,6'-di-O-sinapoyl-sucrose and tenuifolin. This may be because the solubility of 3,6'-di-O-sinapoyl-sucrose and tenuifolin in water is very poor.

[0137] Under the same conditions, when the solvent volume increased from 5 mL to 10 mL, the content of tenuifolin increased slightly, but the content of 3,6'-di-O-sinapoyl-sucrose fluctuated slightly. The optimal solvent addition ratio was determined to be 10 mL of ethanol volume: 1 g of raw material.

[0138] Under the same conditions, drying the water extract can significantly increase the contents of the two active ingredients. The increase multiple can be as high as about 4 times, and the yields of the two active ingredients are both the highest. This may be because the water content in the water extraction precipitate used currently is relatively large. In addition, although the water content in the system after drying + 75 vv% ethanol is higher than that in the system after non-drying + 95 vv% ethanol, the yields of 3,6'-di-O-sinapoyl-sucrose and tenuifolin in the former are still higher than those in the latter. This shows that the process of drying the water extract not only can reduce the water content and increase the extract yield, but also helps the release of 3,6'-di-O-sinapoyl-sucrose and tenuifolin, further increasing the yield. Therefore, in this process, it is preferably to dry the solid part after water extraction and then add 95 vv% ethanol for extraction, so as to achieve the highest extraction yields of 3,6'-di-O-sinapoyl-sucrose and tenuifolin.

[0139] Example 4: Extraction Process of Active Substances from Polygala tenuifolia Willd A co-extraction scheme for active substances from Polygala tenuifolia Willd is as follows: Step 1: Take 1 kg of defatted Polygala tenuifolia Willd stems, soak them in 10 L of water per 1 kg and perform ultrasonic cleaning for 1 - 5 min. Dry them at 60 °C until dry, pulverize them with a pulverizer, and pass through a 100-mesh sieve.

[0140] Step 2: Transfer the above-mentioned Polygala tenuifolia Willd powder to a clean container, add 10 L of drinking water, stir until the powder is completely wetted, and let it stand overnight at 2 - 8 °C.

[0141] Step 3: Filter the mixture through a 5-μm filter membrane under pressure to separate the liquid and solid precipitate.

[0142] Step 3: Transfer the above liquid to a 20-L reaction tank, add 0.15 wt% sodium hydroxide (pH about 10), stir to dissolve, heat to 115 °C for 1 h, add 0.25% citric acid to neutralize to pH = 6, filter through a 100-mesh sieve, and collect the filtrate, which is the water extract.

[0143] Step 4: Dry the solid precipitate obtained in Step 2 above at 60 °C, pulverize it, add 10 L of 95% ethanol, soak it overnight, filter it through a 5-μm filter membrane under pressure, and collect the clarified liquid, which is the ethanol extract.

[0144] Step 5: Combine the above 10 L of water extract and 10 L of ethanol extract, make up to 50 L with purified water, add 5 wt% maltodextrin, and spray-dry it with a spray dryer. The spray-drying conditions are: inlet air temperature 170 °C, feed pressure 0.2 bar, and feed rate 30 rpm / min. Collect the prepared powder, which is the co-extracted powder of active substances from Polygala tenuifolia Willd (about 1000 g), as Sample E4.

[0145] Step 6: Dissolve 1 g of the above powder in 1 ml of methanol, filter it, and detect the contents of total saponins, tenuifolisaponin, 3,4,5-trimethoxycinnamic acid, 3,6'-diferuloylsucrose, and polygalaxanthone III.

[0146] Among them, the yield of total saponins is 3.5%, the yield of tenuifolisaponin is 0.441%, the yield of 3,4,5-trimethoxycinnamic acid is 0.051%, the yield of 3,6'-diferuloylsucrose is 0.22%, and the yield of polygalaxanthone III is 0.084%. The specific data are shown in the following table Table 5: Detection Data of Polygala tenuifolia Willd Powder

[0147]

[0148] Example 5: Efficacy Test of Co-Extracts from Polygala tenuifolia Willd in Improving Sleep in Mice Determine the sleep-improving activity (shortening the pentobarbital sodium sleep latency and prolonging the pentobarbital sodium sleep time) of the polygala tenuifolia powder (purchased from Anhui Kangweifu Pharmaceutical Co., Ltd.), Group C1, Group C2 obtained in Example 1, and the sample of Group E4 obtained in Example 4 Step 1: Administer drugs to mice. After adult female mice are placed in the animal house and adapted to feeding for one week, 50 mice are randomly divided into five groups, with 10 mice in each group. The first group is the blank control group, which is given normal saline (0.2 mL / mouse). The second group is given an aqueous solution of polygala tenuifolia powder (soaked in 5 g / 100 ml of water) (0.2 mL / mouse). The third group is given the sample of Group C1 obtained in Example 1 (dissolved in 5 g / 100 ml of water) (0.2 mL / mouse). The fourth group is given the sample of Group C2 obtained in Example 1 (dissolved in 5 g / 100 mL of water) (0.2 mL / mouse). The fifth group is given the sample of Group E4 obtained in Example 4 (redissolved in 5 g / 100 ml of water) (0.2 mL / mouse). Intragastric administration is performed once a day for 30 consecutive days.

[0149] Step 2: Inject pentobarbital sodium intraperitoneally into each group of animals. The injection dose is 45 mg / kg BW. Using the disappearance of the righting reflex as an index, record the start time of intraperitoneal injection, the sleep onset situation, the sleep onset time, and the waking time of the mice respectively.

[0150] Step 3: Statistically analyze the sleep latency and sleep time of each group of mice. The specific results are as Figure 1 、 2 and shown in Table 6.

[0151] Table 6: Results of the sleep improvement experiment

[0152] It can be seen that Group 5 (i.e., the group administered with the E4 sample) is significantly superior to other control groups in terms of the number of sleeping animals and the sleep onset time, achieving an effect of almost 100% sleep onset and a short sleep onset time. It is also significantly superior to other control groups in terms of the sleep time. It shows that the co-extract of the active components of polygala tenuifolia obtained in the present invention exhibits excellent effects in shortening the pentobarbital sodium sleep latency and prolonging the pentobarbital sodium sleep time.

Claims

1. A co-extract of active ingredients of Polygala tenuifolia Willd., characterized in that, The co-extract of the active components of Polygala tenuifolia Willd. includes active components selected from the following group: tenuifoliside A, polygalaxanthone III, 3,6'-di-O-sinapoyl-sucrose, and 3,4,5-trimethoxycinnamic acid.

2. The co-extract of polygala tenuifolia active ingredients according to claim 1, characterized in that, The content of tenuifoliside A in the co-extract of the active components of Polygala tenuifolia Willd. is ≥ 0.10 mg / mL.

3. The co-extract of polygala tenuifolia active ingredients according to claim 1, characterized in that, The co-extract of the active components of Polygala tenuifolia Willd. also has one or more characteristics selected from the following group: (i) The content of 3,6'-di-O-sinapoyl-sucrose is ≥ 0.05 mg / mL; (ii) The content of 3,4,5-trimethoxycinnamic acid is ≥ 0.01 mg / mL; (ii) The content of polygalaxanthone III is ≥ 0.02 mg / mL.

4. The preparation method of the co-extract of the active ingredients of Polygala tenuifolia Willd. as claimed in claim 1, wherein, The method includes: (1) Providing Polygala tenuifolia Willd. raw materials, drying and pulverizing them to obtain Polygala tenuifolia Willd. powder; (2) Taking the Polygala tenuifolia Willd. powder obtained in step (1), extracting with water to obtain a water extract of Polygala tenuifolia Willd., filtering, and separately collecting the supernatant and the solid part: (3) Taking the supernatant obtained in step (2), adding an alkali solution for reaction, filtering to obtain an alkali-treated filtrate (the first filtrate) and a filter cake; (4) Taking the filter cake obtained in step (3), drying and pulverizing it, adding ethanol with a concentration of 70 - 98 v / v% for extraction, filtering to obtain an alcohol extraction supernatant (the second filtrate) and a filter cake; (5) Combining the alcohol extraction supernatant (the second filtrate) obtained in step (4) and the alkali-treated filtrate (the first filtrate) obtained in step (3) to obtain the co-extract of the active components of Polygala tenuifolia Willd.

5. The preparation method of the co-extract of the active ingredients of Polygala tenuifolia Willd. as claimed in claim 4, wherein, Step (5) also includes: After the alcohol extraction supernatant (the second filtrate) obtained in step (4) and the alkali-treated filtrate (the first filtrate) obtained in step (3) are mixed, adding water for dilution to obtain a dilution solution, and then adding 2 - 10 wt% of maltodextrin to the dilution solution, and spray-drying to obtain the co-extract of the active components of Polygala tenuifolia Willd.

6. The preparation method of the polygala tenuifolia active ingredient co-extract according to claim 4, characterized in that, The concentration of ethanol in step (4) is 90 - 98 v / v%.

7. The preparation method of the co-extract of polygala root active ingredients according to claim 4, characterized in that, The concentration of the alkali solution in step (3) is 0.01 - 0.5 wt%.

8. The preparation method of the co-extract of polygala root active ingredients according to claim 4, characterized in that, The alkali solution in step (3) is sodium hydroxide.

9. The use of the co-extract of the active ingredients of Polygala tenuifolia Willd. as described in claim 1, characterized in that, It is used for preparing a composition for improving sleep.

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

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