An effective part of poria cocos and a preparation method and application thereof

Abstract

The application provides a poria cocos effective part and a preparation method and application thereof, and belongs to the technical field of traditional Chinese medicine extracts. The poria cocos is crushed, alcohol solution is added for cold soaking, and the soaking solution is concentrated under reduced pressure to obtain a extract; the extract is subjected to two times of chromatographic column chromatography to obtain a flow fraction A6-18; the flow fraction A6-18 is left to stand, and the separated solid is the poria cocos effective part. The preparation method of the poria cocos effective part is simple, seven main chemical components are determined, the finally extracted poria cocos effective part (A6-18a) has excellent sedative and hypnotic effects, and has a synergistic effect with sodium pentobarbital.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine extract technology, specifically relating to an effective part of Poria cocos, its preparation method, and its application. Background Technology

[0002] Sleep disorders are a significant health concern, and prolonged insomnia can severely impact daily work and life. Currently, most mainstream medications for insomnia are accompanied by serious side effects, such as melatonin receptor agonists that induce tolerance, benzodiazepines that suppress the central nervous system and produce residual and withdrawal effects, and hypnotics that impair memory. Therefore, finding safe, side-effect-free, and highly effective novel sedative-hypnotic drugs is of paramount importance.

[0003] Poria cocos (Fu Shen) is the sclerotium of the fungus *Poria cocos* containing pine roots (i.e., *Fu Shen wood*). It has a sweet and bland taste, and is neutral in nature. It possesses calming and soothing properties, and promotes urination. It is often used to treat palpitations, insomnia, forgetfulness, epilepsy, and difficulty urinating. Poria cocos is particularly effective in calming the mind and soothing the nerves. Traditional Chinese medicine believes that it is especially effective in treating heart-related illnesses, and modern medicine also uses it to treat mental disorders. As a natural Chinese medicine product that is both food and medicine, Poria cocos has attracted the attention of many researchers in the medical field, becoming an important research subject for treating insomnia. Therefore, further exploring the chemical composition of Poria cocos and finding its pharmacologically effective material basis and effective component group for calming the mind and soothing the nerves is one of the key areas of ongoing research for those skilled in the art. Summary of the Invention

[0004] In view of this, the purpose of the present invention is to provide a method for preparing the effective part of Poria cocos, the prepared effective part of Poria cocos has excellent sedative and hypnotic effects.

[0005] The present invention also aims to provide the application of the prepared effective fraction of Poria cocos in the preparation of drugs for improving sleep.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0007] This invention provides a method for preparing the effective components of Poria cocos, comprising the following steps:

[0008] Poria cocos powder was added to an alcohol solution for cold soaking, and the extract was concentrated under reduced pressure to obtain an extract. The extract was subjected to a first column chromatography to obtain fraction A6. Fraction A6 was subjected to a second column chromatography to obtain fraction A6-18. Fraction A6-18 was allowed to stand, and the solid that precipitated was the effective part of Poria cocos.

[0009] The first column chromatography used 200-300 mesh silica gel column chromatography, and the eluent was dichloromethane-methanol in volume ratios of 1:0, 50:1, 30:1, 20:1, and 15:1, respectively. The eluent fraction with a dichloromethane-methanol volume ratio of 15:1 was collected and was designated as fraction A6.

[0010] The second chromatographic column chromatography used an ODSRP-18 column, with the eluent being a methanol-water solvent consisting of 20% methanol, 30% methanol, 40% methanol, 50% methanol, 60% methanol, 70% methanol, 80% methanol, and 90% methanol in sequence. The elution fraction containing 90% methanol was collected and designated as fraction A6-18.

[0011] Preferably, the alcohol solution is anhydrous ethanol; the amount of alcohol solution added is 2 to 4 times the mass of Poria cocos.

[0012] Preferably, the cold soaking step is repeated 5 to 7 times, each time for 20 to 30 hours.

[0013] Preferably, the elution flow rate of the first column chromatography is 0.08 to 0.15 BV / min.

[0014] Preferably, the elution program for the first column chromatography is: 0–60 min 1:0 dichloromethane-methanol, 60–150 min 50:1 dichloromethane-methanol, 150–240 min 30:1 dichloromethane-methanol, 240–330 min 20:1 dichloromethane-methanol, and 330–420 min 15:1 dichloromethane-methanol.

[0015] Preferably, the elution flow rate of the second column chromatography is 18–22 mL / min.

[0016] Preferably, the elution program for the second column chromatography is as follows: 0–23 min 20% methanol, 23–46 min 30% methanol, 46–69 min 40% methanol, 69–92 min 50% methanol, 92–115 min 60% methanol, 115–138 min 70% methanol, 138–161 min 80% methanol, and 161–184 min 90% methanol.

[0017] The present invention also provides the effective fraction of Poria cocos prepared by the above preparation method.

[0018] The present invention also provides the application of the above-mentioned effective parts of Poria cocos in the preparation of drugs for improving sleep.

[0019] The present invention also provides the combined use of the above-mentioned effective fraction of Poria cocos and sodium pentobarbital in the preparation of a drug for improving sleep.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] The preparation method of the effective part of Poria cocos described in this invention is simple. The effective part of Poria cocos (A6-18a) obtained by extraction has a high retention of effective active ingredients and does not contain ineffective components such as 3β-O-acetyl-16α-hydroxy-lanoster-8,24-diene-21-enoic acid, dehydropachymic acid, and pachymic acid. The purity and proportion of effective active ingredients are high, which effectively improves the sedative and hypnotic effect and is more conducive to guiding the application of Poria cocos in sedation and hypnosis. Attached Figure Description

[0022] Figure 1 Chromatograms of the alcoholic extract of Poria cocos and its reference standard;

[0023] Figure 2 Chromatograms of the alcoholic extract of Poria cocos and the effective fraction of Poria cocos (A6-18a). Detailed Implementation

[0024] This invention provides a method for preparing the effective fraction of Poria cocos, comprising the following steps: pulverizing Poria cocos, adding an alcohol solution for cold soaking, and concentrating the soaking solution under reduced pressure to obtain an extract; performing a first chromatographic column chromatography on the extract to obtain fraction A6; performing a second chromatographic column chromatography on fraction A6 to obtain fraction A6-18; allowing fraction A6-18 to stand, and the precipitated solid is the effective fraction of Poria cocos.

[0025] The Poria cocos sclerotium described in this invention is a Poria cocos sclerotium containing pine roots. This invention involves pulverizing the dried Poria cocos raw material, preferably with a particle size of 10-30 mesh, more preferably 20 mesh.

[0026] This invention involves cold soaking pulverized Poria cocos raw material with an alcohol solution, preferably an anhydrous ethanol solution, in an amount 2-4 times the mass of Poria cocos, preferably 3 times. The cold soaking is preferably performed at room temperature for 20-30 hours, more preferably 24-26 hours. This invention preferably involves 5-7 cold soakings, more preferably 6. As an optional embodiment, after each cold soaking, the extract is collected and the cold soaking step is repeated with an equal amount of alcohol solution. This invention preferably combines the extracts from each cold soaking for use. The final cold-soaked extract is concentrated under reduced pressure until no obvious alcohol odor is detected, and the extract is used for subsequent chromatographic chromatography.

[0027] The first chromatographic column chromatography of this invention preferably uses a 200-300 mesh silica gel column, with an elution flow rate of 0.08-0.15 BV / min, preferably 0.10-0.12 BV / min. The eluent is, in sequence, a dichloromethane-methanol solution with volume ratios of 1:0, 50:1, 30:1, 20:1, 15:1, 10:1, 6:1, 3:1, 1:1, and 0:1. The elution program is as follows: 0–60 min 1:0 dichloromethane-methanol, 60–150 min 50:1 dichloromethane-methanol, 150–240 min 30:1 dichloromethane-methanol, 240–330 min 20:1 dichloromethane-methanol, 330–420 min 15:1 dichloromethane-methanol, 420–510 min 10:1 dichloromethane-methanol, 510–600 min 6:1 dichloromethane-methanol, 600–690 min 3:1 dichloromethane-methanol, 690–780 min 1:1 dichloromethane-methanol, and 780–840 min 0:1 dichloromethane-methanol. This invention collects the elution fraction with a dichloromethane-methanol volume ratio of 15:1, designated as fraction A6.

[0028] The second chromatographic column chromatography described in this invention preferably uses an ODSRP-18 column, with an elution flow rate of 18–22 mL / min, preferably 20 mL / min. The eluent is, sequentially, a methanol-water solvent consisting of 20% methanol, 30% methanol, 40% methanol, 50% methanol, 60% methanol, 70% methanol, 80% methanol, 90% methanol, and 100% methanol. The elution program is: 0–23 min 20% methanol, 23–46 min 30% methanol, 46–69 min 40% methanol, 69–92 min 50% methanol, 92–115 min 60% methanol, 115–138 min 70% methanol, 138–161 min 80% methanol, 161–184 min 90% methanol, and 184–207 min 100% methanol. This invention collects the 90% methanol fraction, designated as fraction A6-18.

[0029] In this invention, the obtained fraction A6-18 is allowed to stand, and the precipitated white solid is the effective component of Poria cocos. Preferably, the precipitated white solid is washed with a 50% methanol solution, and the washed solid is dried to obtain the effective component of Poria cocos.

[0030] This invention also provides the effective fraction (A6-18a) of Poria cocos prepared by the above-mentioned preparation method. Through systematic research and structural identification of the chemical components of Poria cocos using modern spectroscopic identification technology, it was found that the effective fraction of Poria cocos prepared by this invention mainly contains poricoic acid DM, dehydrotumulosic acid, tumulosic acid, dehydrotrametenolic acid, trametenolic acid, dehydroeburicoic acid, and eburicoic acid. It removes ineffective components such as 3β-O-acetyl-16α-hydroxy-lanoster-8,24-diene-21-enoic acid, dehydroporicoic acid, and poricoic acid from Poria cocos, achieving a superior sedative and hypnotic effect compared to Poria cocos and its crude extract.

[0031] The present invention also provides the application of the above-mentioned effective parts of Poria cocos in the preparation of drugs for improving sleep.

[0032] The present invention also provides the combined application of the above-mentioned effective fraction of Poria cocos and sodium pentobarbital in the preparation of a drug for improving sleep, wherein the effective fraction of Poria cocos has a synergistic effect with sodium pentobarbital in prolonging the sleep time of mice.

[0033] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0034] In a specific embodiment of the present invention, the Poria cocos used was collected in Taoling Township, Jinzhai County, Anhui Province in June 2022. Professor Peng Huasheng of the China Academy of Chinese Medical Sciences identified its source as the fungus Poria cocos (Show.) Wolf of the Polyporaceae family, which is a Poria cocos block with pine roots in the middle of the sclerotium.

[0035] In the specific embodiments of the present invention, the instruments and reagents used are as follows: Waters 1525 high-performance liquid chromatograph (Waters Corporation, USA); Waters XBridge C18 series chromatographic columns (19 mm×250 mm, 5 μm or 4.6 mm×150 mm, 5 μm, Waters Corporation, USA); P1050 high-pressure infusion pump (Rehe Technology, China), model: Rehe Technology, 49 mm (ID)*319 mm (L), flow rate 20 mL / min; 200-300 mesh column chromatography silica gel (Qingdao Ocean Chemical Co., Ltd.); thin-layer chromatography silica gel plate (Qingdao Ocean Chemical Co., Ltd.); reversed-phase ODS C-18 silica gel packing material (20-45 mm, Fuji Silysia Chemical Co., Ltd., Japan); Pharmacia Sephadex LH-20 gel material (Amershanm Biosciences Co., Ltd., Sweden); chromatographic methanol and chromatographic acetonitrile (Oceanpak Co., Ltd., UK); deuterated pyridine (Cambridge Isotope Laboratories Co., Ltd., USA); ZZ-6g type mouse spontaneous activity tester (Chengdu Taimeng Technology Co., Ltd.), BS223S type electronic analytical balance (Sartorius Scientific Instruments Co., Ltd., Beijing); black coffee (Nestle (China) Co., Ltd., batch number: 23411125P); PCPA (Sigma Co., Ltd., USA); sodium pentobarbital (Sigma Co., Ltd., USA); mouse gamma-aminobutyric acid ELISA kit (Jiangsu Enzyme Immuno Industry Co., Ltd.); mouse 5-hydroxytryptamine ELISA kit (Jiangsu Enzyme Immuno Industry Co., Ltd.); other reagents are all of analytical grade.

[0036] In the specific embodiments of the present invention, the animals used are male ICR mice, weighing 18-22 g, purchased from Henan Skbeshi Biotechnology Co., Ltd., license number: SCXK (Yu) 2020-000.

[0037] In the specific embodiments of the present invention, SPSS 23.0 software is used for statistical analysis of the data, and the data is adopted is expressed. One-way analysis of variance and LSD-T test are used for inter-group comparison between multiple samples. P<0.05 indicates a significant difference and is statistically significant.

[0038] Example 1

[0039] A preparation method for the effective part of Poria cum Radix Pini, the steps are as follows:

[0040] 10 kg of dried Poria cum Radix Pini medicinal materials are pulverized to 20 mesh, and cold-extracted 6 times with 3 times the amount of absolute ethanol at room temperature for 24 h each time. The extraction solutions are combined and concentrated under reduced pressure until there is no obvious ethanol smell to obtain 145.5 g of extract, and each gram of the sample contains 68.75 g of crude drug content, which is reserved for use.

[0041] 135g of Poria cocos alcohol extract was loaded onto a silica gel column (100-200 mesh) using a wet loading method and a dry loading method. Gradient elution was performed using a dichloromethane-methanol system (1:0, 50:1, 30:1, 20:1, 15:1, 10:1, 6:1, 3:1, 1:1, 0:1, V / V) (flow rate: 0.08-0.15 column volumes (BV) / min, column volume: 950 cm³). 3 Maximum flow rate: 150 mL / min. The elution program was as follows: 0–60 min 1:0 dichloromethane-methanol, 60–150 min 50:1 dichloromethane-methanol, 150–240 min 30:1 dichloromethane-methanol, 240–330 min 20:1 dichloromethane-methanol, 330–420 min 15:1 dichloromethane-methanol, 420–510 min 10:1 dichloromethane-methanol, 510–600 min 6:1 dichloromethane-methanol, 600–690 min 3:1 dichloromethane-methanol, 690–780 min 1:1 dichloromethane-methanol, and 780–840 min 0:1 dichloromethane-methanol. During the elution process, thin-layer chromatography (TLC) was used to identify and combine similar fractions. In the dichloromethane-methanol volume ratio 15:1 fraction, fraction A6 (8.4 g) was obtained.

[0042] Fiber A6 was eluted using an ODSRP-18 column [model: Ruihe Technology, 49mm (ID) * 319mm (L)] at a flow rate of 20 mL / min. The eluent was sequentially 20% methanol, 30% methanol, 40% methanol, 50% methanol, 60% methanol, 70% methanol, 80% methanol, 90% methanol, and 100% methanol in a methanol-water solution. The elution program was: 0–23 min 20% methanol, 23–46 min 30% methanol, 46–69 min 40% methanol, 69–92 min 50% methanol, 92–115 min 60% methanol, 115–138 min 70% methanol, 138–161 min 80% methanol, 161–184 min 90% methanol, and 184–207 min 100% methanol. During the elution process, thin-layer chromatography (TLC) was used to identify and combine similar fractions, and fraction A6-18 was obtained in the 90% methanol fraction.

[0043] After fraction A6-18 was allowed to stand for 10 minutes, a white solid precipitated out. The solid was washed with a solvent (methanol-water, 50:50) to obtain solid A6-18a. After drying, a total of 550 mg was obtained, which yielded a sample containing 17.0 kg of crude drug per gram of sample for later use.

[0044] Example 2

[0045] A method for preparing the effective components of Poria cocos, comprising the following steps:

[0046] 10 kg of dried Poria cocos was pulverized to 30 mesh and soaked in twice the amount of anhydrous ethanol at room temperature seven times, 26 hours each time. The extracts were combined and concentrated under reduced pressure until there was no obvious ethanol odor, yielding 140.2 g of extract. Each gram of sample contained 71.32 g of raw herb, which was then set aside for later use.

[0047] 130g of Poria cocos alcohol extract was loaded onto a silica gel column (100-200 mesh) using a wet loading method and a dry loading method. Gradient elution was performed using a dichloromethane-methanol system (1:0, 50:1, 30:1, 20:1, 15:1, 10:1, 6:1, 3:1, 1:1, 0:1, V / V) (flow rate: 0.1-0.12 column volumes (BV) / min, column volume: 950 cm³). 3 Maximum flow rate: 120 mL / min). The elution procedure was the same as in Example 1. During elution, thin-layer chromatography (TLC) was used for identification, similar fractions were combined, and fraction A6 (8.3 g) was obtained in the 15:1 dichloromethane-methanol volume ratio fraction.

[0048] Fraction A6 was eluted using an ODSRP-18 column [model: Ruihe Technology, 49mm (ID) * 319mm (L)] at a flow rate of 18 mL / min. The eluent was a methanol-water mixture consisting of 20% methanol, 30% methanol, 40% methanol, 50% methanol, 60% methanol, 70% methanol, 80% methanol, 90% methanol, and 100% methanol, in sequence. The elution procedure was the same as in Example 1. During elution, thin-layer chromatography (TLC) was used to identify and combine similar fractions, yielding fraction A6-18 in the 90% methanol fraction.

[0049] After the fraction A6-18 was allowed to stand, a white solid precipitated out. The solid was washed with a solvent (methanol-water, 50:50) to obtain solid A6-18a. After drying, a total of 535 mg was obtained, which yielded a sample containing 17.3 kg of crude drug per gram of sample for later use.

[0050] Example 3

[0051] A method for preparing the effective components of Poria cocos, comprising the following steps:

[0052] 10 kg of dried Poria cocos was pulverized to 20 mesh and soaked five times with four times the amount of anhydrous ethanol at room temperature for 22 hours each time. The extracts were combined and concentrated under reduced pressure until there was no obvious ethanol odor, yielding 138.2 g of extract. Each gram of sample contained 72.4 g of raw herb, which was then set aside for later use.

[0053] 132g of Poria cocos alcohol extract was loaded onto a silica gel column (100-200 mesh) using a wet loading method and a dry loading method. Gradient elution was performed using a dichloromethane-methanol system (0:1, 50:1, 30:1, 20:1, 15:1, 10:1, 6:1, 3:1, 1:1, 0:1, V / V) (flow rate: 0.08-0.15 column volumes (BV) / min, column volume: 950 cm³). 3 Maximum flow rate: 150 mL / min. The elution procedure was the same as in Example 1. During elution, thin-layer chromatography (TLC) was used for identification, similar fractions were combined, and fraction A6 (7.8 g) was obtained in the 15:1 dichloromethane-methanol volume ratio fraction.

[0054] Fraction A6 was eluted using an ODSRP-18 column [model: Ruihe Technology, 49mm (ID) * 319mm (L)] at a flow rate of 22 mL / min. The eluent was a methanol-water mixture consisting of 20% methanol, 30% methanol, 40% methanol, 50% methanol, 60% methanol, 70% methanol, 80% methanol, 90% methanol, and 100% methanol, in sequence. The elution procedure was the same as in Example 1. During elution, thin-layer chromatography (TLC) was used to identify and combine similar fractions, yielding fraction A6-18 in the 90% methanol fraction.

[0055] After the fraction A6-18 was allowed to stand, a white solid precipitated out. The solid was washed with a solvent (methanol-water, 50:50) to obtain solid A6-18a. After drying, a total of 542 mg was obtained, which yielded a sample containing 17.6 kg of crude drug per gram of sample for later use.

[0056] Experimental Example 1

[0057] The components of the extract obtained after alcohol extraction in Example 1 (Poria cocos alcohol extract) and the effective part of Poria cocos (A6-18a) obtained by final extraction were analyzed.

[0058] Preparation of test solution: Accurately weigh 1g of extract (Poria cocos alcohol extract) or 1g of A6-18a (Poria cocos effective part) into a 2ml EP tube, accurately transfer 0.5ml of chromatographic methanol, sonicate until completely dissolved, filter with a 0.45μm organic microporous membrane, and take the filtrate to obtain the Poria cocos alcohol extract or A6-18a test solution.

[0059] Preparation of reference solution: Weigh the following standards separately into EP tubes: pachymucoid DM, pachymucoid B, dehydrotumoric acid, tumoric acid, 3-epi-(3'-hydroxy-3'-methylglutaryloxyl)-dehydrotumulosic acid, 3-epi-dehydropachymucoid, pinicopsic acid A, 3β-O-acetyl-16α-hydroxy-lanosteric-8,24-diene-21-enoic acid, pinocoric acid, dehydropachymucoid, pachymucoid, pinocoric acid, pinocoric acid, dehydrodentioic acid, and dentioic acid. Dissolve them in chromatographic methanol to prepare a reference solution with a concentration of 1 mg / ml.

[0060] Chromatographic analysis was performed using an Agilent 5HC-C18(2) (250 mm × 4.6 mm, 5 μm) column. The chromatographic conditions were as follows: mobile phase: acetonitrile (A) - 0.01% formic acid water (B), gradient elution (0–20 min, 50% A–70% A; 20–40 min, 70% A–100% A; 40–45 min, 100% A–100% A; 45–50 min, 50% A–50% A), flow rate: 1.0 mL / min, detection wavelength: 200 nm, column temperature: 40 °C, and injection volume: 10 μl.

[0061] Chromatograms of Poria cocos alcohol extract and reference standards are shown below. Figure 1 . Figure 1 The crude extract of Poria cocos is the alcoholic extract of Poria cocos. Figure 1The following are listed in Chinese: 1. poricoic acid DM, 2. poricoic acid B, 3. dehydrotumulosic acid, 4. tumulosic acid, 5. 3-epi-(3'-hydroxy-3'-methylglutaryloxyl)-dehydrotumulosic acid, 6. 3-epi-dehydropachymic acid, 7. pinicopsic acid A, 8. 3β-O-acetyl-16α-hydroxy-lanosta-8,24-dien-21-oic acid, 9. dehydropachymic acid, 10. pachymic acid, 11. dehydrotrametenolic acid, 12. trametenolic acid. 13. Dehydroeburicoic acid, 14. Eburicoic acid. The structures of each component are as follows:

[0062]

[0063] Chromatograms of the alcoholic extract of Poria cocos and the effective components of Poria cocos are shown below. Figure 2 . Figure 2 The crude extract of Poria cocos is the alcoholic extract of Poria cocos, and A6-18a is the effective part of Poria cocos. Figure 2The following are listed in Chinese: 1. poricoic acid DM, 2. poricoic acid B, 3. dehydrotumulosic acid, 4. tumulosic acid, 5. 3-epi-(3'-hydroxy-3'-methylglutaryloxyl)-dehydrotumulosic acid, 6. 3-epi-dehydropachymic acid, 7. pinicopsic acid A, 8. 3β-O-acetyl-16α-hydroxy-lanosta-8,24-dien-21-oic acid, 9. dehydropachymic acid, 10. pachymic acid, 11. dehydrotrametenolic acid, 12. trametenolic acid. acid, 13. dehydroeburicoic acid, 14. eburicoic acid.

[0064] According to chromatogram Figures 1-2 Peak 11 (with a moderate peak area and good separation) was selected as the reference peak, and the relative peak areas and peak contents of other peaks were calculated. The calculated peak areas and peak contents of each component in the ethanol extract of Poria cocos and the effective fraction of Poria cocos (A6-18a) are shown in Tables 1-2.

[0065] Table 1. Relative peak areas and peak contents of peaks 1-14 of the Poria cocos alcohol extract.

[0066]

[0067] Table 2. Relative peak areas and peak contents of peaks 1, 3, 4, and 11-14 of the effective fraction of Poria cocos (A6-18a).

[0068]

[0069] Comparing the components of the ethanol extract of Poria cocos and the effective part of Poria cocos (A6-18a), it can be seen that peak 8 (3β-O-acetyl-16α-hydroxy-lanoster-8,24-diene-21-enoic acid), peak 9 (dehydropachymic acid), and peak 10 (pachymic acid) are the main differences between the ethanol extract of Poria cocos and the effective part of Poria cocos (A6-18a), and their content is higher in the ethanol extract of Poria cocos.

[0070] Experimental Example 2

[0071] The extract obtained after alcohol extraction in Example 1 (Poria cocos alcohol extract) and the effective part of Poria cocos (A6-18a) were used to investigate the sedative and hypnotic effects of Poria cocos and its effective part on mice.

[0072] 1. Effects on spontaneous activity in normal mice

[0073] Forty-eight ICR mice were randomly divided into six groups (n=6-8 per group): a normal control group, a medium-dose group of Poria cocos extract (20 g / kg crude drug, i.e., 286 mg / kg sample), a high-dose group of Poria cocos extract (40 g / kg crude drug, i.e., 572 mg / kg sample), a low-dose group of Poria cocos A6-18a (20 g / kg crude drug, i.e., 1.15 mg / kg sample), a medium-dose group of Poria cocos A6-18a (60 g / kg crude drug, i.e., 3.45 mg / kg sample), and a high-dose group of Poria cocos A6-18a (200 g / kg crude drug, i.e., 11.5 mg / kg sample). All groups were administered the drug at the prescribed dosage, with an administration volume of 0.2 mL / 10 g. The normal control group received an equal volume of distilled water. The administration was performed once daily for six consecutive days. On day 5, 45 minutes after administration, the mice were placed in a self-acclimatization device for 5 minutes. The mice were fasted for 12 hours before the last administration, but allowed free access to water. On the 6th day, 45 minutes after administration, the mice were placed in a spontaneous activity device for 1 minute to acclimatize. The number of times the mice moved and stood in the device within 5 minutes was recorded. The results are shown in Table 3.

[0074] Table 3. Effects of spontaneous activity on normal mice in each group (n = 6–8, )

[0075]

[0076] Compared with the normal group: #P<0.05, ##P<0.01, ###P<0.001

[0077] As shown in Table 3, compared with the normal group, the number of spontaneous activities in the high-dose group of Poria cocos extract was significantly reduced (P<0.01), and the number of spontaneous activities in the high-dose group of Poria cocos A6-18a was significantly reduced (P<0.001). This suggests that both the Poria cocos extract and the effective part of Poria cocos (A6-18a) have a certain sedative effect on mice, with the effective part of Poria cocos (A6-18a) having a more significant effect.

[0078] 2. Effects of caffeine on the excitatory effect in mice

[0079] Fifty-six ICR mice were randomly divided into seven groups of six to eight mice each: normal control group, model group, medium-dose group of Poria cocos extract (crude drug 20 g / kg, i.e., sample amount 286 mg / kg), high-dose group of Poria cocos extract (crude drug 40 g / kg, i.e., sample amount 572 mg / kg), low-dose group of Poria cocos A6-18a (crude drug 20 g / kg, i.e., sample amount 1.15 mg / kg), medium-dose group of Poria cocos A6-18a (crude drug 60 g / kg, i.e., sample amount 3.45 mg / kg), and high-dose group of Poria cocos A6-18a (…). The crude drug concentration was 200 g / kg (i.e., sample amount 11.5 mg / kg). Each treatment group was administered the drug at the prescribed dose, with an administration volume of 0.2 mL / 10 g. The normal control group received an equal volume of distilled water, and the drug was mixed with coffee (1 g / kg) at a 2:1 volume ratio and administered by gavage. The model group received a 2:1 dilution of water and coffee, administered by gavage at a volume of 0.3 mL / 10 g. Administration continued for 3 days. Mice were fasted but allowed free water on the first night of administration. On the third day, 30 minutes after administration, the mice were placed in a self-adaptation device for 1 minute. Their activity within the device was recorded for 5 minutes. The results are shown in Table 4.

[0080] Table 4. Effects of caffeine-induced excitation on spontaneous activity in mice of different groups (n = 6–8). )

[0081]

[0082] Compared with the normal group: #P<0.05, ##P<0.01, ###P<0.001; Compared with the model group: *P<0.05, **P<0.01, ***P<0.001

[0083] Table 4 shows that, compared with the normal group, the model group had a significantly increased number of spontaneous activities and standing frequency (P<0.001), indicating that coffee (1g / kg) has an excitatory effect on mice. Compared with the model group, the high-dose group of Poria cocos extract had a significantly decreased number of spontaneous activities (P<0.05), and the medium-dose and high-dose groups of Poria cocos A6-18a also had significantly decreased numbers of spontaneous activities (P<0.01). The high-dose group of Poria cocos extract also had a significantly decreased number of standing frequency (P<0.01), and the high-dose group of Poria cocos A6-18a also had a significantly decreased number of standing frequency (P<0.05). This indicates that the effective components of Poria cocos can antagonize the central nervous system excitatory effect of coffee, suggesting that the ethanol extract of Poria cocos and the effective component (A6-18a) have a certain sedative effect on coffee-excited mice, with the effective component (A6-18a) showing a more significant effect.

[0084] 3. Effects of PCPA on PCPA-induced insomnia in mice

[0085] Fifty-six ICR mice were randomly divided into seven groups of six to eight mice each: normal control group, model group, medium-dose group of Poria cocos extract (20 g / kg crude drug, i.e., 286 mg / kg sample), high-dose group of Poria cocos extract (40 g / kg crude drug, i.e., 572 mg / kg sample), low-dose group of Poria cocos A6-18a (20 g / kg crude drug, i.e., 1.15 mg / kg sample), and medium-dose group of Poria cocos A6-18a. The mice were administered drugs at the following doses: 60 g / kg crude drug (equivalent to 3.45 mg / kg sample); 200 g / kg crude drug (equivalent to 11.5 mg / kg sample); and 0.2 mL / 10 g sample. The normal control group received an equal volume of distilled water via gavage once daily. All groups except the normal control group received intraperitoneal injections of PCPA (350 mg / kg) for three consecutive days. The normal control group received an equal volume of physiological saline intraperitoneally. Thirty minutes after administration on the third day, the mice were placed in a self-adaptation device for 1 minute, and their activity within the device was recorded for 5 minutes. The results are shown in Table 5.

[0086] Table 5. Effects of PCPA-induced insomnia on spontaneous activity in mice of different groups (n = 6–8). )

[0087]

[0088] Compared with the normal group: #P<0.05, ##P<0.01, ###P<0.001; Compared with the model group: *P<0.05, **P<0.01, ***P<0.001

[0089] Table 5 shows that, compared with the normal group, the number of activities in the model group was significantly increased (P<0.01), suggesting that PCPA (350mg / kg) has an insomnia-inducing effect on mice. Compared with the model group, the number of spontaneous activities and standing times in the high-dose group of Poria cocos A6-18a was significantly reduced (P<0.05), suggesting that the effective part of Poria cocos (A6-18a) has a certain hypnotic effect on PCPA-induced insomnia in mice.

[0090] On the third day after PCPA discontinuation (during the PCPA discontinuation period, mice were continuously administered Poria cocos extract or Poria cocos A6-18a via gavage), and 45 minutes after administration, mice in each group were intraperitoneally injected with a suprathreshold dose of sodium pentobarbital (46 mg / kg). The sleep latency and sleep duration were recorded (the criterion for sleep onset was the disappearance of the righting reflex for more than 1 minute; the criterion for sleep onset was the number of times the mouse turned over 3 times within 1 minute; the criterion for sleep termination was the number of times the mouse turned over 3 times within 1 minute; the recovery time was recorded). The sleep latency and sleep duration were calculated. The results are shown in Table 6.

[0091] Table 6. Effects of PCPA-induced insomnia in mice of different groups on synergistic sleep with suprathreshold doses of sodium pentobarbital (n = 6–8). )

[0092]

[0093] Compared with the normal group: #P<0.05, ##P<0.01, ###P<0.001; Compared with the model group: *P<0.05, **P<0.01, ***P<0.001

[0094] Table 6 shows that, compared with the normal group, the sleep time of mice in the model group was significantly prolonged after injection of the suprathreshold dose of sodium pentobarbital (P<0.001). Compared with the model group, the medium and high dose groups of Poria cocos A6-18a significantly prolonged the sleep time of mice injected with the suprathreshold dose of sodium pentobarbital (P<0.01), and the high dose group of Poria cocos alcohol extract also prolonged the sleep time of mice injected with the suprathreshold dose of sodium pentobarbital, but the difference was not significant; the results suggest that the effective part of Poria cocos (A6-18a) has a synergistic effect with sodium pentobarbital in prolonging the sleep time of mice.

[0095] 4. Measurement of GABA in the hypothalamus and 5-HT in the brainstem of mice

[0096] (1) Preparation of tissue samples

[0097] In step 3, one hour after the last administration of the drug to the mice, the eyeballs were enucleated and blood was collected after anesthesia. Then, brain tissue from the hypothalamus and brainstem was quickly removed and frozen in liquid nitrogen. Before testing, the tissue was weighed and PBS was quickly added in a glass homogenizer at a ratio of 1:9 (tissue:PBS buffer) and thoroughly homogenized. The homogenate was then centrifuged at 4°C and 3000 rpm for 20 min, and the supernatant was collected and frozen for later testing.

[0098] (2) Experimental methods

[0099] Refer to the kit instructions for specific procedures: Dilute the corresponding γ-GABA and 5-HT standards to the required concentrations to prepare standard stock solutions. Set up blank wells, standard wells, and sample wells on the enzyme-labeled plate. Do not add sample or enzyme-labeled reagent to the blank wells. Add 50 μL of standard to the standard wells. Add 40 μL of sample diluent to the sample wells, followed by 10 μL of the sample to be tested. Cover with the sealing film, gently shake, and incubate at 37°C for 60 min. Remove the sealing film, shake dry, fill each well with diluted washing buffer, let stand for 30 s, discard, and repeat 5 times. Pat dry. Add 50 μL of enzyme-labeled reagent to each well, except for the blank wells, and repeat the incubation and washing steps. Pat dry, add 50 μL of chromogenic reagent A to each well, followed by 50 μL of chromogenic reagent B. Incubate at 37°C in the dark for 10 min, add 50 μL of stop solution, and measure the absorbance at 450 nm within 15 min. The effects of each group on the levels of GABA in the hypothalamus and 5-HT in the brainstem of mice were calculated, and the results are shown in Table 7.

[0100] Table 7. Effects of each group on the levels of GABA and 5-HT in the hypothalamus and brainstem of mice (n = 6–8). )

[0101]

[0102] Compared with the normal group: #P<0.05, ##P<0.01, ###P<0.001; Compared with the model group: *P<0.05, **P<0.01, ***P<0.001

[0103] Table 7 shows that, compared with the normal group, the hypothalamic GABA content in the model group was significantly decreased (P<0.05). Compared with the model group, GABA levels in both the high-dose group of Poria cocos extract and the high-dose group of Poria cocos A6-18a were increased, but the difference was not statistically significant, suggesting that the ethanol extract of Poria cocos and the effective part of Poria cocos (A6-18a) may exert a sedative effect by enhancing the neuroinhibitory effect of GABA. Compared with the normal group, the hypothalamic 5-HT content in the model group was significantly decreased (P<0.01). Compared with the model group, 5-HT levels in both the high-dose group of Poria cocos extract and the high-dose group of Poria cocos A6-18a were significantly increased (P<0.05-0.01), indicating that the model was successfully established, and the ethanol extract of Poria cocos and the effective part of Poria cocos (A6-18a) may exert a sedative effect by affecting 5-HT metabolism in the brain.

[0104] In summary, the experimental results showed that both the effective fraction of Poria cocos (A6-18a) and the ethanol extract of Poria cocos significantly reduced spontaneous activity in normal mice and caffeine-induced stimulated mice, and significantly prolonged the sleep time of PCPA-induced insomnia mice injected with a suprathreshold dose of sodium pentobarbital. Furthermore, the effective fraction of Poria cocos (A6-18a) was more effective than the ethanol extract. 5-HT is widely distributed in the central nervous system, participates in the regulation of various mental activities, and can trigger sleep. This experiment, using a classic insomnia model, found that the effective fraction of Poria cocos (A6-18a) (50 g / kg) and the ethanol extract of Poria cocos (40 g / kg) could increase the levels of 5-HT and GABA in the brain tissue of PCPA-induced insomnia mice, and significantly improved their sleep status. Both the ethanol extract of Poria cocos and the effective part of Poria cocos (A6-18a) have sedative and hypnotic effects, and the effective part of Poria cocos (A6-18a) is more effective than the ethanol extract. This indicates that the effective part of Poria cocos (A6-18a) finally extracted by the method described in this invention removes some ineffective components. Compared with previous studies on the sedative and hypnotic pharmacological material basis of Poria cocos, this invention, through a two-step preparation method, clarifies the pharmacological material basis of the sedative and hypnotic effects of Poria cocos and improves the purity and content of the effective components, which can effectively improve the sedative and hypnotic effects.

[0105] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing the effective components of Poria cocos, characterized in that, Includes the following steps: Poria cocos powder was added to an alcohol solution for cold soaking, and the extract was concentrated under reduced pressure to obtain an extract. The extract was subjected to a first column chromatography to obtain fraction A6. Fraction A6 was subjected to a second column chromatography to obtain fraction A6-18. Fraction A6-18 was allowed to stand, and the solid that precipitated was the effective part of Poria cocos. The first column chromatography used 200-300 mesh silica gel column chromatography, and the eluent was dichloromethane-methanol in volume ratios of 1:0, 50:1, 30:1, 20:1, and 15:1, respectively. The eluent fraction with a dichloromethane-methanol volume ratio of 15:1 was collected and was designated as fraction A6. The second chromatographic column chromatography used an ODS RP-18 column, with the eluent being a methanol-water solvent consisting of 20% methanol, 30% methanol, 40% methanol, 50% methanol, 60% methanol, 70% methanol, 80% methanol, and 90% methanol in sequence. The elution fraction containing 90% methanol was collected and designated as fraction A6-18.

2. The preparation method according to claim 1, characterized in that, The alcohol solution is anhydrous ethanol; the amount of alcohol solution added is 2 to 4 times the mass of Poria cocos.

3. The preparation method according to claim 1, characterized in that, The cold soaking step is repeated 5 to 7 times, each time for 20 to 30 hours.

4. The preparation method according to claim 1, characterized in that, The elution flow rate for the first column chromatography was 0.08–0.15 BV / min.

5. The preparation method according to claim 1, characterized in that, The elution program for the first column chromatography was as follows: 0–60 min 1:0 dichloromethane-methanol, 60–150 min 50:1 dichloromethane-methanol, 150–240 min 30:1 dichloromethane-methanol, 240–330 min 20:1 dichloromethane-methanol, and 330–420 min 15:1 dichloromethane-methanol.

6. The preparation method according to claim 1, characterized in that, The elution flow rate for the second column chromatography was 18–22 mL / min.

7. The preparation method according to claim 1, characterized in that, The elution program for the second column chromatography was as follows: 0–23 min 20% methanol, 23–46 min 30% methanol, 46–69 min 40% methanol, 69–92 min 50% methanol, 92–115 min 60% methanol, 115–138 min 70% methanol, 138–161 min 80% methanol, and 161–184 min 90% methanol.

8. The effective fraction of Poria cocos prepared by the preparation method according to any one of claims 1 to 7.

9. The use of the effective component of Poria cocos as described in claim 8 in the preparation of a drug for improving sleep.

10. The combined use of the effective fraction of Poria cocos as described in claim 8 and sodium pentobarbital in the preparation of a drug for improving sleep.

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