An improved insomnia-improving qimen an tea polysaccharide ANTP-1 and a preparation method thereof

Abstract

The application discloses Qimen Ancha polysaccharide ANTP-1 for improving insomnia and a preparation method thereof, and belongs to the field of separation and purification of plant active ingredients. The Qimen Ancha polysaccharide ANTP-1 is composed of mannose, rhamnose, glucose, galactose, xylose and arabinose, and the molar ratio is 1.00:0.36:16.78:1.96:4.50:8.21, and the relative molecular weight is 26.58 kDa. The Qimen Ancha polysaccharide ANTP-1 can significantly shorten the sleep latency of sleep-deprived mice, prolong the sleep duration, and effectively improve insomnia. The Qimen Ancha polysaccharide ANTP-1 has a clear composition, remarkable efficacy, and a broad development and application prospect in sleep aid.

Description

Technical Field

[0001] This invention belongs to the field of plant active ingredient isolation and purification, specifically relating to a Qimen tea polysaccharide ANTP-1 for improving insomnia and its preparation method. Background Technology

[0002] Insomnia is a common sleep disorder, typically characterized by difficulty falling asleep, maintaining sleep, or early awakening, accompanied by significant daytime functional impairment. Insomnia not only affects quality of life but is also closely related to the development of various chronic diseases such as depression, anxiety, cardiovascular disease, metabolic syndrome, and immune dysfunction, seriously threatening people's daily lives and physical health. Current mainstream treatments rely on benzodiazepines and melatonin, but these interventions only provide short-term symptom relief, and long-term use can easily lead to drug dependence and metabolic burden. Therefore, developing functional ingredients that are naturally derived, highly safe, and possess broad pharmacological activity to construct long-term management strategies for insomnia has significant research value and promising clinical application prospects.

[0003] Qimen An-Tea, produced in Qimen County, Anhui Province, China, is a traditional Chinese post-fermented compressed tea. Its tea leaves are tightly rolled, dark in color, with a rich and lasting aroma, and a bright liquor. It possesses significant aging value and potential health benefits. In long-term folk consumption, An-Tea has been believed to have a "calming and soothing" effect, suggesting potential application value in improving sleep. Tea polysaccharides, as one of the key active components of An-Tea, have been reported to possess diverse and significant biological activities. However, current research on An-Tea remains relatively weak, especially systematic studies on the composition and function of An-Tea polysaccharides, which are almost nonexistent. This limits the in-depth understanding and development of An-Tea's biological activities. Therefore, analyzing the main active components of Qimen An-Tea polysaccharides and evaluating their physiological functions is of great significance for scientifically explaining the traditional effects of An-Tea and exploring its modern health applications. Summary of the Invention

[0004] To address the shortcomings of the existing technology, this invention provides a Qimen tea polysaccharide ANTP-1 for improving insomnia and its preparation method. This invention isolates a highly active heteropolysaccharide from Qimen tea, which significantly improves sleep disorders in sleep-deprived mice, exhibiting excellent sleep-aiding activity and serving as an ideal candidate for sleep aid.

[0005] The present invention relates to Qimen Ancha polysaccharide, abbreviated as ANTP-1, with a relative molecular weight of 26.58 kDa. The monosaccharide composition and molar ratio are mannose:rhamnose:glucose:galactose:xylose:arabinose = 1.00:0.36:16.78:1.96:4.50:8.21.

[0006] The preparation method of Qimen Ancha polysaccharide of the present invention includes the following steps:

[0007] Step 1: Finely grind the dried Qimen An tea, pass it through an 80-mesh sieve, remove fat-soluble impurities and pigments by reflux with ethanol, and dry it at 60 ℃ for 24 hours to obtain Qimen An tea powder;

[0008] Step 2: Add the Qimen An tea powder obtained in Step 1 to distilled water at a material-to-liquid ratio of 1:20 (g / ml), extract at 80 ℃ for 2 hours, repeat the extraction 3 times, filter through 8 layers of gauze, and combine the extracts;

[0009] Step 3: Concentrate the extract obtained in Step 2 to 1 / 20 of the original volume, add 4 times the volume of 95% ethanol, stir thoroughly, let stand at 4 ℃ for 8-12 h, centrifuge at 4000 rpm for 10 min, and collect the precipitate.

[0010] Step 4: The precipitate obtained in Step 3 was subjected to protein removal by the Sevag method, dialyzed, and freeze-dried to obtain crude polysaccharide from Qimen tea.

[0011] Step 5: The crude polysaccharide of Qimen Ancha obtained in Step 4 was separated and purified using a DEAE-Sepharose Fast Flow ion exchange chromatography column. The components were eluted with deionized water, collected in separate tubes, concentrated, and freeze-dried to obtain Qimen Ancha polysaccharide ANTP-1.

[0012] This invention utilizes a DEAE-Sepharose Fast Flow chromatographic column to rapidly prepare Qimen Ancha polysaccharide ANTP-1, which has a molecular weight of 26.58 kDa and is composed of mannose, rhamnose, glucose, galactose, xylose, and arabinose.

[0013] This invention relates to the application of Qimen Ancha polysaccharide in the preparation of food, health products, and pharmaceutical formulations.

[0014] The food, health products, and pharmaceutical preparations mentioned above have significant sleep-aiding effects. Attached Figure Description

[0015] Figure 1 The HPLC chromatogram of ANTP-1, a polysaccharide from Qimen Ancha tea.

[0016] Figure 2 The diagram shows the monosaccharide composition of Qimen Ancha polysaccharide ANTP-1, where A is the peak elution diagram of standard monosaccharides and B is the peak elution diagram of ANTP-1 monosaccharides.

[0017] Figure 3 The results of the open field experiment are shown in Figure A, where A is the trajectory diagram, B is the statistical diagram of the distance traveled by mice in each group to enter the central area, and C is the statistical diagram of the total distance traveled by mice in each group.

[0018] Figure 4 The diagram shows the results of the sodium pentobarbital sleep experiment, where A and B represent the sleep latency and total sleep duration of mice in each group, respectively.

[0019] Figure 5 The study investigated the effects of Qimen Ancha polysaccharide ANTP-1 on neurotransmitter levels in the hypothalamus of insomnia model mice. A, B, C, and D represent the levels of 5-hydroxytryptamine (5-HT), γ-aminobutyric acid (GABA), norepinephrine (NE), and dopamine (DA), respectively. Detailed Implementation

[0020] The preparation and sleep-aiding activity of the present invention are described below with reference to specific embodiments and accompanying drawings. The listed embodiments are only used to explain the present invention and are not intended to limit the scope of protection of the present invention.

[0021] Example 1: Preparation of Qimen tea polysaccharide ANTP-1

[0022] Step 1: Finely grind the dried Qimen Ancha tea, pass it through an 80-mesh sieve, and vacuum filter it. Remove fat-soluble impurities and pigments by reflux treatment with ethanol at 78-82℃ for 3-4 hours, and then dry it at 60℃ for 24 hours to obtain Qimen Ancha powder.

[0023] Step 2: Add the Qimen An tea powder obtained in Step 1 to distilled water at a material-to-liquid ratio of 1:20 (w / v), extract at 80 ℃ for 2 hours, repeat the extraction 3 times, filter through 8 layers of gauze, and combine the extracts.

[0024] Step 3: Concentrate the extract obtained in Step 2 to 1 / 20 of its original volume, add 4 times the volume of 95% ethanol, stir thoroughly, let stand at 4 ℃ for 8-12 h, centrifuge at 4000 rpm for 10 min, and collect the precipitate.

[0025] Step 4: The precipitate obtained in Step 3 was deproteinized using the Sevag method, dialyzed through a 3500 Da dialysis bag, and freeze-dried to obtain the crude polysaccharide ANTP from Qimen Ancha tea.

[0026] Step 5: The crude polysaccharide of Qimen Ancha obtained in Step 4 was separated and purified using a DEAE-Sepharose Fast Flow ion exchange chromatography column. The components were eluted with deionized water, collected in separate tubes, concentrated, and freeze-dried to obtain Qimen Ancha polysaccharide ANTP-1.

[0027] Example 2: Molecular weight determination and monosaccharide composition of Qimen tea polysaccharide ANTP-1

[0028] (1) Molecular weight determination

[0029] ANTP-1 and dextran standards (T10, T50, T500, T1000, T2000) were prepared into 2 mg / mL solutions with double-distilled water and analyzed using an Agilent high-performance liquid chromatography-evaporative light scattering detector (HPLC-ELSD). The chromatographic column was a TSK GelG6000 PWXL, the mobile phase was deionized water, the carrier gas was N2, the gas flow rate was 2.5 L / min, and the injection volume was 10 μL. A standard curve was plotted using the logarithm of the relative molecular mass (Lg Mw) and retention time (Rt) of the standards, and the molecular weight range of ANTP-1 was determined. The results are as follows: Figure 1 As shown. The molecular weight of ANTP-1 was calculated to be 26.58 kDa based on the standard curve.

[0030] (2) Monosaccharide composition analysis

[0031] The monosaccharide composition of the sample after ANTP-1 acid hydrolysis-pre-column PMP (1-phenyl-3-methyl-5-pyrazolone) derivatization was determined using an Agilent high-performance liquid chromatography combined with a diode array detector (DAD). 10 mg of ANTP-1 was dissolved in 5 mL of 2 mol / L trifluoroacetic acid. After sealing the tube with nitrogen, it was incubated in an oil bath at 110 °C for 8 h. Methanol was added repeatedly and the solution was rotary evaporated until the pH reached 7. 1 mL of deionized water was added for later use. 1 mL each of 5 mg / mL standard monosaccharide solution and acid-hydrolyzed ANTP-1 solution were added to NaOH (0.5 mL, 0.3 mol / L) and PMP working solution (0.5 mL, 0.5 mol / L), respectively. After shaking and mixing, the solution was reacted in a water bath at 70 °C for 1 h for PMP pre-column derivatization. The solution was then neutralized to neutral with HCl (0.5 mL, 0.3 mol / L). Excess PMP was removed by extraction three times with chloroform. The aqueous phase was filtered through a 0.22 μm filter membrane and detected by HPLC-DAD.

[0032] HPLC-DAD conditions: The column was a Zorbox Eclipse XDB-C18 column, the column temperature was 30 ℃, and the wavelength was 245 nm; the mobile phase A was acetonitrile, the mobile phase B was 0.05 mol / L phosphate buffer solution, gradient elution was performed from 0 to 60 min, the initial setting was mobile phase A: mobile phase B = 17%: 83%, and the final elution ratio was mobile phase A: mobile phase B = 20%: 80%; the injection volume was 10 uL.

[0033] The results are as follows Figure 2 As shown, A is the peak chromatogram of the standard monosaccharide, and B is the peak chromatogram of the ANTP-1 monosaccharide; from Figure 2It can be known from [reference] that ANTP-1 is composed of 6 kinds of monosaccharides, and the monosaccharide composition and molar percentage are mannose:rhamnose:glucose:galactose:xylose:arabinose = 1.00:0.36:16.78:1.96:4.50:8.21.

[0034] Example 3: Hypnotic Activity of Qimen Ancha Polysaccharide ANTP-1

[0035] Using the prepared Qimen Ancha polysaccharide ANTP-1 as the raw material, a sleep-deprived mouse model was established using a sleep deprivation instrument to evaluate the hypnotic effect of Qimen Ancha polysaccharide ANTP-1.

[0036] (1)Construction of Sleep-Deprived Mouse Model

[0037] Forty-eight SPF-grade C57BL / 6 mice (male, 6 - 8 weeks old, 18 - 22 g) were selected and provided by Hefei Qingyuan Biotechnology Co., Ltd. The animal license number is: SCXK (Zhe) 2024 - 0004. After all mice were adaptively fed, the mice were randomly divided into 6 groups (n = 8), namely the normal group (NC), the sleep deprivation group (IM), the melatonin positive drug group (MT), the ANTP-1 low-dose group (LANTP-1), the ANTP-1 medium-dose group (MANTP-1), and the ANTP-1 high-dose group (HANTP-1). The normal group was not treated. Mice in the other groups were sleep-deprived for 20 hours every day for 1 week using a chronic sleep deprivation instrument (rotation speed of 5 rpm). The sleep deprivation started at 20:00 every day and continued until 16:00 the next day, for a total of 20 hours. From the 8th day, the normal group and the sleep deprivation group were gavaged with an equal volume of normal saline, and the other groups were gavaged with 5 mg / kg melatonin, 50 mg / kg polysaccharide ANTP-1, 100 mg / kg polysaccharide ANTP-1, and 200 mg / kg polysaccharide ANTP-1, once a day for 2 weeks. After the last administration on the 21st day, a pentobarbital sodium sleep experiment was conducted, and an open field experiment was conducted on the 22nd day. After the experiment, the mice were anesthetized and sacrificed, and blood and brain tissue samples were collected to measure the levels of 5-HT, GABA, NE, and DA in the hypothalamus tissues of each group. The obtained data were analyzed for significance using GraphPad Prism 9.5 software.

[0038] (2)Experimental Results

[0039] As Figure 3 shown in A, compared with the normal group, the number of times the model group mice passed through the central grid was significantly reduced, indicating that insomniac mice had anxiety behaviors. Figure 3 B and Figure 3Data from C showed that, compared with the model group, the ANTP-1 treatment groups (LANTP-1, MANTP-1, HANTP-1) exhibited significantly increased motor distance into the central region and total distance of activity, showing a certain dose-dependent effect. Among them, HANTP-1 treatment showed the best effect, which was superior to that of the MT group, indicating that ANTP-1 can effectively alleviate anxiety-like behaviors induced by insomnia.

[0040] like Figure 4 As shown, compared with the normal group, the sleep latency of mice in the model group was significantly prolonged, while the sleep duration was significantly shortened. ANTP-1 intervention significantly prolonged the sleep duration and shortened the sleep latency in mice, and this improvement was dose-dependent, indicating that ANTP-1 can effectively improve sleep quality and demonstrates clear sleep-aiding potential.

[0041] like Figure 5 As shown, compared with the normal group, the hypothalamic homogenate levels of 5-HT and GABA in the model group mice were significantly decreased, while the levels of NE and DA were significantly increased. Compared with the model group, the ANTP-1 intervention group significantly increased the levels of 5-HT and GABA in the hypothalamic homogenate of mice, and significantly decreased the levels of NE and DA. Among them, the high-dose HANTP-1 group had returned to normal levels. This indicates that ANTP-1 can improve sleep by regulating the balance between excitatory (NE, DA) and inhibitory (5-HT, GABA) neurotransmitters in the hypothalamus.

Claims

1. A polysaccharide from Qimen tea, abbreviated as ANTP-1, characterized in that: The relative molecular weight of the Qimen Ancha polysaccharide is 26.58 kDa, and the monosaccharide composition and molar ratio are mannose:rhamnose:glucose:galactose:xylose:arabinose = 1.00:0.36:16.78:1.96:4.50:8.

21.

2. The method for preparing Qimen Ancha polysaccharide according to claim 1, characterized in that... Includes the following steps: Step 1: Finely grind the dried Qimen An tea, pass it through an 80-mesh sieve, remove fat-soluble impurities and pigments by reflux with ethanol, and then dry it to obtain Qimen An tea powder; Step 2: Add the Qimen An tea powder obtained in Step 1 to distilled water, extract at 80 ℃ for 2 hours, repeat the extraction 3 times, filter through 8 layers of gauze, and combine the extracts; Step 3: Concentrate the extract obtained in Step 2 to 1 / 20 of its original volume, add 4 times the volume of 95% ethanol, stir thoroughly, let stand at 4 ℃ for 8-12 h, and collect the precipitate by centrifugation; Step 4: The precipitate obtained in Step 3 was subjected to protein removal by the Sevag method, dialyzed, and freeze-dried to obtain crude polysaccharide from Qimen tea. Step 5: The crude polysaccharide of Qimen Ancha obtained in Step 4 was separated, purified, concentrated, and freeze-dried using a DEAE-Sepharose Fast Flow ion exchange chromatography column to obtain Qimen Ancha polysaccharide ANTP-1.

3. The preparation method according to claim 2, characterized in that: In step 2, Qimen An tea powder and distilled water are mixed at a ratio of 1:

20.

4. The preparation method according to claim 2, characterized in that: In step 5, when using a DEAE-Sepharose Fast Flow ion exchange column for separation and purification, deionized water is used for elution.

5. The use of the Qimen Ancha polysaccharide according to claim 1 in the preparation of food and / or health products with sleep-aiding effects.

6. The use of the Qimen Ancha polysaccharide according to claim 1 in the preparation of a pharmaceutical preparation with sleep-aiding effects.

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