Hypericum perforatum polysaccharide with efficacy of preventing and treating depression, and preparation method and application thereof

Abstract

The present application relates to a kind of Hypericum perforatum polysaccharide with the efficacy of preventing and treating depression, the weight average molecular weight (Mw) of the Hypericum perforatum polysaccharide is 200Da-70000Da.The Hypericum perforatum polysaccharide of the present application can be used to prevent and treat depression and anti-fatigue, etc., and has the advantages of high bioavailability, safe and effective, etc.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biological medicine, and particularly relates to a Hypericum perforatum L. polysaccharide with the efficacy of preventing and treating depression, a preparation method thereof and application thereof. BACKGROUND

[0002] According to the investigation and statistics of the World Health Organization (WHO) in 2012, there are about 350 million patients with depression in the world. The annual prevalence rate of depression is 1.5%, and the lifetime prevalence rate is 3.1%. The annual prevalence rate of major depression in the United States is about 7%, in Europe is 2%-5%, in Africa is 1%-7%, and in China is about 2.1%. The incidence of depression in patients with Parkinson's disease is 25.5%-70%, the incidence of depression in patients with stroke is 30%-64%, and the incidence of depression in patients with myocardial infarction is about 45%. Depression has become the second largest disease burden in the world after coronary heart disease.

[0003] Depression affects the mental health of patients and causes various damages, and its common symptoms include low mood, restlessness, unhappiness, anxiousness, inattention, and unclear thinking. The treatment drugs for depression include traditional Chinese medicines (such as modified Chaihu Shugan Powder and modified Jiawei Xiaoyao Powder), Chinese patent medicines (such as Shugan Jieyu Granules or Capsules, Jiawei Xiaoyao Pills, Chaihu Shugan Pills, Renshen Jianpi Pills, Zhibai Dihuang Pills, etc.), and western medicines (such as fluvoxetine and duloxetine), and generally need long-term medication, and the tolerance and safety need to be improved.

[0004] Hypericum perforatum (alias Hypericum japonicum, St. John's wort) is the dry aboveground part of Hypericum perforatum L. of Guttiferae. The alcohol extract of Hypericum perforatum has been used as an antidepressant. St. John's wort extract tablets (Serafem ®The Hypericum perforatum dry extract 300 mg (including Hypericin content not less than 9 mg and total hypericin content not less than 0.4 mg) is used for depression, anxiety and / or restlessness. The Hypericum perforatum extract is prepared by extracting Hypericum perforatum with 50-80% (v / v) ethanol solution or methanol solution. The Shuganjieyu capsule is prepared by extracting Hypericum perforatum 1800 g and Acanthopanax 1500 g, has the effects of soothing liver and relieving depression, invigorating spleen and tranquilizing mind, and is used for treating mild and moderate single-phase depression with liver depression and spleen deficiency, and symptoms such as low mood, decreased interest, stagnation, insomnia, nervousness, irritability, poor appetite, chest tightness, weakness, sweating, pain and the like. The Hypericum perforatum is extracted twice by reflux extraction with 70% (v / v) ethanol solution for one hour each time, the extract is combined, filtered, concentrated under reduced pressure to the extract with a relative density of about 1.10 (70 DEG C), and then spray dried to obtain dry extract powder. The Hypericum perforatum alcohol extraction residue needs to be treated as waste, and the Hypericum perforatum polysaccharides (HPP) contained in the residue are not fully utilized. Studies have shown that the Hypericum perforatum polysaccharides have the effects of improving brain blood and oxygen supply, promoting growth and development, improving damaged or low immune function after radiotherapy and chemotherapy, and resisting viruses, and the anti-depression effect has not been reported. Therefore, it is necessary to comprehensively utilize the Hypericum perforatum extraction residue and fully develop the effects of the Hypericum perforatum polysaccharides, so as to realize maximum utilization of resources and meet the clinical needs. SUMMARY

[0005] The present application aims to provide a Hypericum perforatum polysaccharide with the effect of preventing and treating depression, wherein the weight average molecular weight (Mw) of the Hypericum perforatum polysaccharide is 200 Da-70000 Da, preferably 250 Da-68000 Da.

[0006] In the preferred technical solution of the present application, the Hypericum perforatum polysaccharide contains 20-80% neutral sugar, 10-70% uronic acid and 5-40% protein by weight percentage.

[0007] In the preferred technical solution of the present application, the Hypericum perforatum polysaccharide contains 30-50% neutral sugar, 50-70% uronic acid and 20-30% protein by weight percentage.

[0008] In the preferred technical solution of the present application, the Hypericum perforatum polysaccharide contains 44.3% neutral sugar, 58.6% uronic acid and 26.4% protein by weight percentage.

[0009] In the preferred technical scheme of the present application, the neutral sugar, the uronic acid and the protein contained in the Hypericum perforatum L. polysaccharide are 41.7%, 62.6% and 28.7% respectively in percentage by weight.

[0010] In the preferred technical scheme of the present application, the neutral sugar, the uronic acid and the protein contained in the Hypericum perforatum L. polysaccharide are 41.7%, 62.6% and 28.7% respectively in percentage by weight.

[0011] In the preferred technical scheme of the present application, the molar ratio of arabinose: glucose: galactose: mannose: galacturonic acid in the Hypericum perforatum L. polysaccharide is 1:0.1-10:0.1-10:0.1-10:0.1-10.

[0012] In the preferred technical scheme of the present application, the molar ratio of arabinose: glucose: galactose: mannose: galacturonic acid in the Hypericum perforatum L. polysaccharide is 1:1-10:1-5:0.1-1:0.1-5.

[0013] In the preferred technical scheme of the present application, the molar ratio of arabinose: glucose: rhamnose: galactose: mannose in the Hypericum perforatum L. polysaccharide is 1:0.1-10:0.1-10:0.1-10:0.1-10.

[0014] In the preferred technical scheme of the present application, the molar ratio of arabinose: glucose: rhamnose: galactose: mannose in the Hypericum perforatum L. polysaccharide is 1:1-5:0.1-1:1-5:0.1-1.

[0015] In the preferred technical scheme of the present application, the molar ratio of arabinose: glucose: galactose: mannose: galacturonic acid in the Hypericum perforatum L. polysaccharide is 1.00:1.42:1.41:0.19:0.57.

[0016] In the preferred technical scheme of the present application, the molar ratio of arabinose: glucose: rhamnose: galactose: mannose in the Hypericum perforatum L. polysaccharide is 1.00:2.57:0.25:1.52:0.36.

[0017] In the preferred technical scheme of the present application, the molar ratio of arabinose: glucose: galactose: mannose: galacturonic acid in the Hypericum perforatum L. polysaccharide is 1.00:9.03:2.44:0.75:2.32.

[0018] In the preferred technical scheme of the present application, the preparation of the Hypericum perforatum L. polysaccharide comprises the following steps:

[0019] (1) water is added to the Hypericum perforatum L. in a solid-liquid ratio of 1:1-15, and soaked for 0.5-4 h, and then extracted at a temperature of 90-100 ℃ for 0.5-5 h, and then filtered, and the filtrate and the residue are collected;

[0020] (2) according to the extraction method of step (1), the filter residue is extracted repeatedly for 1-3 times, and the filtrate is collected;

[0021] (3) the filtrates collected in steps (1)-(2) are combined, centrifuged (2000-4000 rpm) * (5 min-30 min), and the collected supernatant is concentrated under reduced pressure to a crude drug concentration of 1:0.5-5 (m / v). After cooling to room temperature, 95% ethanol solution is added to the concentrated solution at a volume ratio of 1:1-5, and the mixture is allowed to stand for 24-72 h. Then, the mixture is centrifuged (2000-4000 rpm) * (5 min-30 min), and the supernatant and precipitate are taken;

[0022] (4) according to the mass / volume ratio of crude drug material: water of 1:1-10, water is added to the collected precipitate in step (3), and the mixture is stirred and dissolved (100-1000 rpm) * (5 min-30 min). Then, the mixture is centrifuged (2000-4000 rpm) * (5 min-30 min), and the supernatant and precipitate are collected;

[0023] (5) referring to the purification method of step (4), the collected precipitate in step (4) is purified repeatedly for 1-4 times;

[0024] (6) the supernatants collected in steps (4)-(5) are combined, and the mixture is concentrated under reduced pressure at 50-80°C to a crude drug concentration of 0.5-2 (m / v). Then, the mixture is dried to obtain the product;

[0025] Alternatively, the preparation of Hypericum perforatum polysaccharide comprises the following steps:

[0026] S-1: according to the solid-liquid ratio of 1:1-15, 50-80% ethanol solution is added to Hypericum perforatum, and the mixture is soaked for 0.5-4 h. Then, the mixture is heated to 65-100°C, and refluxed for 0.5-5 h. After filtration, the residue is collected;

[0027] S-2: according to the extraction method of step S-1, the collected residue is refluxed for 1-2 times, and then filtered to collect the residue;

[0028] S-3: according to the solid-liquid ratio of 1:1-15, water is added to the collected residue, and the mixture is heated to 90-100°C for extraction for 0.5-5 h. After filtration, the filtrate and residue are collected;

[0029] S-4: according to the extraction method of step S-3, the residue is extracted repeatedly for 1-4 times, and the filtrate is collected;

[0030] S-5: combine the filtrate collected in steps S-3 to S-4, centrifuge (2000-4000 rpm) * (5-30 min), and concentrate the collected supernatant under reduced pressure to a crude drug concentration of 1:0.5-5 (m / v). After cooling to room temperature, add 95% ethanol solution to the concentrated solution at a volume ratio of 1:1-5, and stand for 24-72 h. Then centrifuge (2000-4000 rpm) * (5-30 min), and take the supernatant and precipitate;

[0031] S-6: add water to the precipitate collected in step S-5 at a mass / volume ratio of 1:1-10, and stir to dissolve (100-1000 rpm) * (5-30 min). Then centrifuge (2000-4000 rpm) * (5-30 min), and collect the supernatant and precipitate;

[0032] S-7: repeat the purification of the collected precipitate 1-4 times according to the purification method of step S-6;

[0033] S-8: combine the supernatants collected in steps S-6 to S-7, and concentrate under reduced pressure at 50-80°C to a crude drug concentration of 0.5-2 (m / v). Then dry to obtain the product.

[0034] In a preferred technical solution of the present application, the ratio of material to liquid in step (1), S-1 or S-3 is 1:4-12, preferably 1:5-10.

[0035] In a preferred technical solution of the present application, the soaking time in step (1) or S-1 is 1-3 h.

[0036] In a preferred technical solution of the present application, the concentration of the ethanol solution in step S-1 is 60-70%.

[0037] In a preferred technical solution of the present application, the extraction time in steps (1), (2), S-1, S-2, S-3 or S-4 is 1-3 h.

[0038] In a preferred technical solution of the present application, the number of times of repeated extraction of the filter residue in step (2) or S-4 is 2-3 times.

[0039] In a preferred technical solution of the present application, the crude drug concentration in steps (3), (6), S-5 or S-8 is 1-2 (m / v).

[0040] In a preferred technical solution of the present application, the volume ratio of concentrated solution to 95% ethanol in step (3) or S-5 is 1:1-3.

[0041] In a preferred technical solution of the present application, the mass / volume ratio of crude drug to water in step (4) or S-6 is 1:5-8.

[0042] In the preferred technical scheme of the present application, the purification times of the precipitate in step (5) or S-7 is 2-3 times.

[0043] In the preferred technical scheme of the present application, the drying in step (6) or S-8 is selected from any one or combination of reduced pressure drying, spray drying and freeze drying.

[0044] Another object of the present application is to provide a preparation method of Hypericum perforatum L. polysaccharide with the efficacy of preventing and treating depression, wherein the weight average molecular weight (Mw) of the Hypericum perforatum L. polysaccharide is 200 Da-70000 Da, and the method comprises the following steps:

[0045] (1) water is added to Hypericum perforatum L. in a solid-liquid ratio of 1:1-15, and soaked for 0.5-4 h, and then extracted at a temperature of 90-100 ℃ for 0.5-5 h, and then filtered to collect the filtrate and the residue;

[0046] (2) the residue is extracted repeatedly for 1-3 times according to the extraction method of step (1), and the filtrate is collected;

[0047] (3) the filtrates collected in steps (1)-(2) are combined, centrifuged (2000-4000 rpm) * (5 min-30 min), and the collected supernatant is reduced pressure concentrated to a crude drug concentration of 1:0.5-5 (m / v), and then cooled to room temperature, and then 95% ethanol solution is added to the concentrated solution in a volume ratio of 1:1-5, and then placed for 24-72 h, and then centrifuged (2000-4000 rpm) * (5 min-30 min), and then the supernatant and the precipitate are taken;

[0048] (4) water is added to the precipitate collected in step (3) in a mass-volume ratio of 1:1-10, and then stirred and dissolved (100-1000 rpm) * (5 min-30 min), and then centrifuged (2000-4000 rpm) * (5 min-30 min), and then the supernatant and the precipitate are collected;

[0049] (5) the precipitate collected in step (4) is purified repeatedly for 1-4 times according to the purification method of step (4);

[0050] (6) the supernatants collected in steps (4)-(5) are combined, and then placed at 50-80 ℃ for reduced pressure concentration to a crude drug concentration of 0.5-2 (m / v), and then dried to obtain the product;

[0051] Alternatively, the preparation of Hypericum perforatum L. polysaccharide comprises the following steps:

[0052] S-1: according to the material liquid ratio 1:1-15, 50-80% ethanol solution is added to Hypericum perforatum, soaked for 0.5-4h, heated to 65-100℃, refluxed for 0.5-5h, filtered, and the residue was collected;

[0053] S-2: according to the extraction method of step S-1, the collected residue was refluxed for 1-2 times, filtered, and the residue was collected;

[0054] S-3: according to the material liquid ratio 1:1-15, water was added to the collected residue, heated to 90-100℃, extracted for 0.5-5h, filtered, and the filtrate and residue were collected;

[0055] S-4: according to the extraction method of step S-3, the residue was repeatedly extracted for 1-4 times, and the filtrate was collected;

[0056] S-5: the filtrate collected in steps S-3 to S-4 was combined, centrifuged (2000-4000rpm) * (5min-30min), the collected supernatant was reduced pressure concentrated to the crude drug concentration of 1:0.5-5 (m / v), cooled to room temperature, 95% ethanol solution was added to the concentrated solution according to the volume ratio of concentrated solution:95% ethanol solution 1:1-5, and then placed for 24-72h, centrifuged (2000-4000rpm) * (5min-30min), and the supernatant and precipitate were taken;

[0057] S-6: according to the mass volume ratio of crude drug material:water 1:1-10, water was added to the collected precipitate in step S-5, stirred and dissolved (100-1000rpm) * (5min-30min), centrifuged (2000-4000rpm) * (5min-30min), and the supernatant and precipitate were collected;

[0058] S-7: according to the purification method of step S-6, the collected precipitate was repeatedly purified for 1-4 times;

[0059] S-8: the supernatant collected in steps S-6 to S-7 was combined, and placed at 50-80℃ for reduced pressure concentration to the crude drug concentration of 0.5-2 (m / v), and dried to obtain.

[0060] In the preferred technical scheme of the present application, the material liquid ratio in step (1), S-1 or S-3 is 1:4-12, preferably 1:5-10.

[0061] In the preferred technical scheme of the present application, the soaking time in step (1) or S-1 is 1-3h.

[0062] In the preferred technical scheme of the present application, the concentration of the ethanol solution in step S-1 is 60-70%.

[0063] In the preferred technical scheme of the present application, the extraction time in step (1), (2), S-1, S-2, S-3 or S-4 is 1-3h.

[0064] In the preferred technical scheme of the present application, the number of repeated extraction of the filter residue in step (2) or S-4 is 2-3 times.

[0065] In the preferred technical scheme of the present application, the concentration of crude drug in step (3), (6), S-5 or S-8 is 1-2 (m / v).

[0066] In the preferred technical scheme of the present application, the volume ratio of concentrated solution to 95% ethanol in step (3) or S-5 is 1:1-3.

[0067] In the preferred technical scheme of the present application, the mass-volume ratio of crude medicinal material to water in step (4) or S-6 is 1:5-8.

[0068] In the preferred technical scheme of the present application, the number of purification of the precipitate in step (5) or S-7 is 2-3 times.

[0069] In the preferred technical scheme of the present application, the drying in step (6) or S-8 is selected from any one or combination of reduced pressure drying, spray drying and freeze drying.

[0070] In the preferred technical scheme of the present application, the weight average molecular weight (Mw) of the Hypericum perforatum L. polysaccharide is 250Da-68000Da.

[0071] In the preferred technical scheme of the present application, the Hypericum perforatum L. polysaccharide contains 20-80% of neutral sugar, 10-70% of uronic acid and 5-40% of protein by weight percentage.

[0072] In the preferred technical scheme of the present application, the Hypericum perforatum L. polysaccharide contains 30-50% of neutral sugar, 50-70% of uronic acid and 20-30% of protein by weight percentage.

[0073] In the preferred technical scheme of the present application, the Hypericum perforatum L. polysaccharide contains 44.3% of neutral sugar, 58.6% of uronic acid and 26.4% of protein by weight percentage.

[0074] In the preferred technical scheme of the present application, the Hypericum perforatum L. polysaccharide contains 41.7% of neutral sugar, 62.6% of uronic acid and 28.7% of protein by weight percentage.

[0075] In the preferred technical scheme of the present application, the Hypericum perforatum L. polysaccharide contains 35.6% of neutral sugar, 57.4% of uronic acid and 26.0% of protein by weight percentage.

[0076] In the preferred technical solution of the present application, the molar ratio of arabinose: glucose: galactose: mannose: galacturonic acid in the Hypericum perforatum L. polysaccharide is 1:0.1-10:0.1-10:0.1-10:0.1-10.

[0077] In the preferred technical solution of the present application, the molar ratio of arabinose: glucose: galactose: mannose: galacturonic acid in the Hypericum perforatum L. polysaccharide is 1:1-10:1-5:0.1-1:0.1-5.

[0078] In the preferred technical solution of the present application, the molar ratio of arabinose: glucose: rhamnose: galactose: mannose in the Hypericum perforatum L. polysaccharide is 1:0.1-10:0.1-10:0.1-10:0.1-10.

[0079] In the preferred technical solution of the present application, the molar ratio of arabinose: glucose: rhamnose: galactose: mannose in the Hypericum perforatum L. polysaccharide is 1:1-5:0.1-1:1-5:0.1-1.

[0080] In the preferred technical solution of the present application, the molar ratio of arabinose: glucose: galactose: mannose: galacturonic acid in the Hypericum perforatum L. polysaccharide is 1.00:1.42:1.41:0.19:0.57.

[0081] In the preferred technical solution of the present application, the molar ratio of arabinose: glucose: rhamnose: galactose: mannose in the Hypericum perforatum L. polysaccharide is 1.00:2.57:0.25:1.52:0.36.

[0082] In the preferred technical solution of the present application, the molar ratio of arabinose: glucose: galactose: mannose: galacturonic acid in the Hypericum perforatum L. polysaccharide is 1.00:9.03:2.44:0.75:2.32.

[0083] Another object of the present application is to provide a Hypericum perforatum L. polysaccharide composition having the efficacy of preventing and treating depression, which is composed of the Hypericum perforatum L. polysaccharide of the present application and a pharmaceutically acceptable carrier, wherein the weight average molecular weight (Mw) of the Hypericum perforatum L. polysaccharide is 200 Da-70000 Da.

[0084] In the preferred technical solution of the present application, the weight average molecular weight (Mw) of the Hypericum perforatum L. polysaccharide is 250 Da-68000 Da.

[0085] In the preferred technical solution of the present application, the Hypericum perforatum L. polysaccharide contains 20-80% of neutral sugar, 10-70% of uronic acid and 5-40% of protein in terms of weight percentage.

[0086] In the preferred technical solution of the present application, the Hypericum perforatum polysaccharide contains 30-50% of neutral sugar, 50-70% of uronic acid and 20-30% of protein by weight percentage.

[0087] In the preferred technical solution of the present application, the Hypericum perforatum polysaccharide contains 44.3% of neutral sugar, 58.6% of uronic acid and 26.4% of protein by weight percentage.

[0088] In the preferred technical solution of the present application, the Hypericum perforatum polysaccharide contains 41.7% of neutral sugar, 62.6% of uronic acid and 28.7% of protein by weight percentage.

[0089] In the preferred technical solution of the present application, the Hypericum perforatum polysaccharide contains 35.6% of neutral sugar, 57.4% of uronic acid and 26.0% of protein by weight percentage.

[0090] In the preferred technical solution of the present application, the molar ratio of arabinose: glucose: galactose: mannose: galacturonic acid in the Hypericum perforatum polysaccharide is 1:0.1-10:0.1-10:0.1-10:0.1-10.

[0091] In the preferred technical solution of the present application, the molar ratio of arabinose: glucose: galactose: mannose: galacturonic acid in the Hypericum perforatum polysaccharide is 1:1-10:1-5:0.1-1:0.1-5.

[0092] In the preferred technical solution of the present application, the molar ratio of arabinose: glucose: rhamnose: galactose: mannose in the Hypericum perforatum polysaccharide is 1:0.1-10:0.1-10:0.1-10:0.1-10.

[0093] In the preferred technical solution of the present application, the molar ratio of arabinose: glucose: rhamnose: galactose: mannose in the Hypericum perforatum polysaccharide is 1:1-5:0.1-1:1-5:0.1-1.

[0094] In the preferred technical solution of the present application, the molar ratio of arabinose: glucose: galactose: mannose: galacturonic acid in the Hypericum perforatum polysaccharide is 1.00:1.42:1.41:0.19:0.57.

[0095] In the preferred technical solution of the present application, the molar ratio of arabinose: glucose: rhamnose: galactose: mannose in the Hypericum perforatum polysaccharide is 1.00:2.57:0.25:1.52:0.36.

[0096] In the preferred technical scheme of the present application, the molar ratio of arabinose: glucose: galactose: mannose: galacturonic acid in the Hypericum perforatum L. polysaccharide is 1.00:9.03:2.44:0.75:2.32.

[0097] In the preferred technical scheme of the present application, the amount or type of the pharmaceutically acceptable carrier in the pharmaceutical composition is determined according to the physicochemical properties and content of the active ingredient, the type of preparation, the dissolution and bioavailability of the preparation and other factors.

[0098] In the preferred technical scheme of the present application, the dosage form of the pharmaceutical composition is oral preparation.

[0099] In the preferred technical scheme of the present application, the oral preparation is selected from any one of oral liquid preparation, tablet, capsule, granule, paste, syrup, powder, lotion, effervescent, suspension, pill, dripping pill, mixture, paste, emulsion, liniment.

[0100] In the preferred technical scheme of the present application, the pharmaceutically acceptable carrier of the present application is a common excipient or adjuvant used in the art for preparing the desired preparation, including filler (diluent), disintegrant, binder, lubricant (glidant or anti-adhesion agent), dispersant, humectant, pH regulator (acid-base regulator), osmotic pressure regulator, pore-forming agent, solubilizer, antioxidant, bacteriostatic agent (bactericide), analgesic agent (anesthetic), suspending agent, emulsifier, co-emulsifier, lyophilization protective agent, flavoring agent, odorant, etc.

[0101] In the preferred technical scheme of the present application, the filler is selected from any one or combination of lactose, sugar powder, dextrin, starch, sodium carboxymethyl starch, sodium starch glycolate, pregelatinized starch, modified starch, hydroxypropyl starch, potato starch, corn starch, cellulose, inorganic calcium salt, calcium chloride, calcium sulfate, calcium phosphate, calcium hydrogen phosphate, precipitated calcium carbonate, sorbitol, mannitol, microcrystalline cellulose, sodium carboxymethyl cellulose, ethyl cellulose, hydroxypropyl methyl cellulose.

[0102] In the preferred technical scheme of the present application, the disintegrant is selected from any one or combination of starch, sodium carboxymethyl starch, sodium starch glycolate, pregelatinized starch, modified starch, hydroxypropyl starch, potato starch, corn starch or its derivative, cross-linked polyvinyl pyrrolidone, microcrystalline cellulose, low-substituted hydroxypropyl cellulose, cross-linked sodium carboxymethyl cellulose.

[0103] In the preferred technical solution of the present application, the adhesive is selected from any one or combination of sugar syrup, gum arabic, gelatin, sorbitol, tragacanth gum, cellulose, microcrystalline cellulose, sodium carboxymethyl cellulose, ethyl cellulose, methyl cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, gelatin syrup, starch syrup, polyvinylpyrrolidone, sodium carboxymethyl starch, sodium starch glycolate, pregelatinized starch, modified starch, hydroxypropyl starch, potato starch, corn starch.

[0104] In the preferred technical solution of the present application, the lubricant is selected from any one or combination of micronized silica gel, magnesium stearate, talc, colloidal silicon dioxide, aluminum hydroxide, boric acid, hydrogenated vegetable oil, polyethylene glycol, sodium dodecyl sulfate.

[0105] In the preferred technical solution of the present application, the wetting agent is selected from any one or combination of sodium dodecyl sulfate, polysorbate (Tween), water, alcohol, ester.

[0106] In the preferred technical solution of the present application, the antioxidant is selected from any one or combination of sodium sulfite, sodium bisulfite, sodium pyrosulfite, sodium thiosulfate, vitamin C, vitamin E, dibutyl phenyl acid, butyl hydroxy anisole (BHA), butyl hydroxy toluene (BHT), propyl gallate (PG), nicotinamide, acetylcysteine, tertiary butyl hydroquinone (TBHQ).

[0107] In the preferred technical solution of the present application, the bacteriostatic agent (bactericide) is selected from any one or combination of phenol, cresol, chlorobutanol, benzyl alcohol.

[0108] In the preferred technical solution of the present application, the analgesic agent (anesthetic) is selected from any one or combination of chlorobutanol, benzyl alcohol, lidocaine, procaine.

[0109] In the preferred technical solution of the present application, the suspending agent is selected from any one or combination of microcrystalline cellulose, sodium carboxymethyl cellulose, ethyl cellulose, methyl cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, sugar syrup, glycerol, gelatin, gum arabic, gum tragacanth, sodium alginate, potassium alginate, pectin.

[0110] In the preferred technical solution of the present application, the acid-base regulator (pH regulator) is selected from any one or combination of hydrochloric acid, sodium hydroxide, potassium hydroxide, citric acid, sodium citrate, potassium citrate, malic acid, sodium malate, potassium malate, sodium phosphate monobasic, disodium hydrogen phosphate, calcium hydroxide, calcium lactate, sodium lactate, sodium phosphate, potassium phosphate, dipotassium hydrogen phosphate, potassium phosphate monobasic, sodium bicarbonate, sodium carbonate.

[0111] In the preferred technical solution of the present application, the osmotic pressure regulator is selected from any one of sodium chloride, potassium chloride, glucose, phosphate, citrate.

[0112] In the preferred technical solution of the present application, the emulsifier is selected from any one or combination of sodium stearate, potassium stearate, stearic acid triethanolamine, magnesium stearate, calcium stearate, sodium dodecyl sulfate, sodium dodecyl benzene sulfonate, polysorbate (Tween), Span, Sijiaz, Benzaze, polyvinyl alcohol, tragacanth gum, acacia gum, pluronic F-68, lecithin, and soybean phospholipid.

[0113] In the preferred technical solution of the present application, the co-emulsifier is selected from any one or combination of n-butanol, ethylene glycol, ethanol, propylene glycol, glycerol, and polyglycerol ester.

[0114] In the preferred technical solution of the present application, the lyophilization protective agent is selected from any one or combination of sucrose, lactose, galactose, glucose, trehalose, mannitol, and sorbitol.

[0115] In the preferred technical solution of the present application, the solubilizing agent is selected from any one or combination of Tween-80, pluronic F-68, Sijiaz, Benzaze, cholic acid salt, deoxycholic acid salt, glycerol, propylene glycol, and polyethylene glycol.

[0116] In the preferred technical solution of the present application, the flavoring agent is selected from any one or combination of honey, syrup, essence, and sweetener.

[0117] In the preferred technical solution of the present application, the Hypericum perforatum L. polysaccharide is prepared into a sustained-release preparation or controlled-release preparation according to the method in the art by adding a retardant coating or preparing a matrix-type preparation, etc.

[0118] In the preferred technical solution of the present application, the sustained-release preparation carrier or controlled-release preparation carrier is selected from any one or combination of oleaginous incorporation agent, hydrophilic colloid, water-insoluble retardant, enteric retardant, and biodegradable retardant.

[0119] In the preferred technical solution of the present application, the oleaginous incorporation agent is selected from any one or combination of glycerol monostearate, hydrogenated castor oil, mineral oil, polysiloxane, and dimethyl silicone.

[0120] In the preferred technical solution of the present application, the hydrophilic colloid is selected from any one or combination of methyl cellulose (MC), sodium carboxymethyl cellulose (CMC-Na), hydroxyethyl cellulose (HEC), hydroxypropyl cellulose (HPC), hydroxypropyl methyl cellulose (HMPC), polyvinylpyrrolidone (PVP), acacia gum, tragacanth gum, or carboxymethylcellulose, polyvinyl alcohol (PVA), pectin, alginate, chitosan, xanthan gum, guar gum, furcelleran gum, gelatin, agar, and galactomannan.

[0121] The water-insoluble retardant is preferably selected from ethyl cellulose (EC), cellulose acetate (CA), polyethylene, polypropylene, polysiloxane, ethylene-vinyl acetate copolymer (EVA), polymethyl methacrylate, or a combination thereof.

[0122] The enteric retardant is preferably selected from cellulose acetate phthalate (CAP), hydroxypropyl methylcellulose phthalate (HMPCP), polyvinyl alcohol phthalate (PVAP), hydroxypropyl methylcellulose acetate succinate (HPMCAS), acrylic resin, or a combination thereof.

[0123] The biodegradable retardant is preferably selected from waxes, fatty acids, fatty acid esters, fatty alcohols, carnauba wax, stearic acid, glyceryl monostearate, stearyl alcohol, cetyl alcohol, or a combination thereof.

[0124] The drug composition is preferably administered orally, mucosally, or dermally.

[0125] Another object of the present application is to provide the use of the Hypericum perforatum L. polysaccharide or the composition thereof in the preparation of a product for preventing and treating depression.

[0126] Another object of the present application is to provide the use of the Hypericum perforatum L. polysaccharide or the composition thereof in the preparation of an anti-fatigue product.

[0127] Unless otherwise specified, the following terms are defined as follows in the present application:

[0128] 1. The ratio of material to liquid in the present application refers to the mass / volume ratio (m / v) of crude medicinal materials and extraction solvent;

[0129] 2. The crude drug concentration in the present application refers to the mass / volume ratio (m / v) of crude medicinal materials and concentrated solution;

[0130] 3. The abbreviations of the terms in the present application: 1-phenyl-3-methyl-5-pyrazolone (PMP); xylose (Xyl); arabinose (Ara); glucose (Glu); rhamnose (Rha); fucose (Fuc); galactose (Gal); mannose (Man); glucuronic acid (GluUA); galacturonic acid (GalUA).

[0131] Unless otherwise specified, the percentage between liquids is volume / volume percentage; the percentage between a liquid and a solid is volume / weight percentage; the percentage between a solid and a liquid is weight / volume percentage; and the rest is weight / weight percentage.

[0132] The present application adopts the following method for determination:

[0133] 1. The present application adopts the phenol-sulfuric acid method to determine the neutral sugar content (calculated as glucose); adopts the m-hydroxydiphenyl method to determine the uronic acid content (calculated as glucuronic acid); and adopts the Coomassie brilliant blue method to determine the protein content (calculated as bovine serum albumin).

[0134] 2. The present application completely acid hydrolyzes the Hypericum perforatum L. polysaccharide and performs PMP derivatization, and adopts the capillary electrophoresis method (Capillary electrophoresis, CE) to determine the monosaccharide composition and molar ratio thereof.

[0135] Instrument: American Beckman Company; buffer salt solution: 50 mmol / L borax solution; capillary column: Φ50 μm*60 cm; separation voltage: 15 kV; detection wavelength: 245 nm; sample injection pressure: 0.5 psi; sample injection time: 10 s; column temperature: 25 DEG C.

[0136] 3. The present application adopts the high-performance gel-permeation chromatography method (High-performance gel-permeation chromatography, HPGPC) to determine the molecular weight distribution of the polysaccharide.

[0137] Instrument: HPLC, Waters Company; chromatographic column: TSKsw2500 or 3000; mobile phase: 0.1 M Na2SO4; flow rate: 0.6 mL / min; detector: differential.

[0138] 4. An appropriate amount of Hypericum perforatum L. polysaccharide is mixed with KBr, grinded uniformly, and a high-throughput Fourier infrared (FT-IR) spectrometer is used to detect and record the infrared spectrum thereof.

[0139] Compared with the prior art, the present application has the following beneficial technical effects:

[0140] 1. The present application scientifically screens the extraction method of Hypericum perforatum L. polysaccharide, and the prepared Hypericum perforatum L. polysaccharide has excellent anti-depression, anti-fatigue and other effects, and has the advantages of high bioavailability, safety and effectiveness.

[0141] 2. The preparation method of the present application has the advantages of simple operation, high yield, more optimal cost, green environmental protection, and suitability for industrialized production. BRIEF DESCRIPTION OF DRAWINGS

[0142] Figure 1 The CE spectrum of the Hypericum perforatum L. polysaccharide prepared in Example 1 of the present application is shown in the following figure:

[0143] Figure 2The HPGPC spectrum of the Hypericum perforatum L. polysaccharide prepared in Example 1 is obtained;

[0144] Figure 3 The FT-IR spectrum of the Hypericum perforatum L. polysaccharide prepared in Example 1 is obtained;

[0145] Figure 4 The CE spectrum of the Hypericum perforatum L. polysaccharide prepared in Example 2 is obtained;

[0146] Figure 5 The HPGPC spectrum of the Hypericum perforatum L. polysaccharide prepared in Example 2 is obtained;

[0147] Figure 6 The FT-IR spectrum of the Hypericum perforatum L. polysaccharide prepared in Example 2 is obtained;

[0148] Figure 7 The CE spectrum of the Hypericum perforatum L. polysaccharide prepared in Example 3 is obtained;

[0149] Figure 8 The HPGPC spectrum of the Hypericum perforatum L. polysaccharide prepared in Example 3 is obtained;

[0150] Figure 9 The FT-IR spectrum of the Hypericum perforatum L. polysaccharide prepared in Example 3 is obtained;

[0151] Figure 10 The influence of the Hypericum perforatum L. polysaccharide on the body weight of each group of mice before and after administration;

[0152] Figure 11 The influence of the Hypericum perforatum L. polysaccharide (low-dose group, medium-dose group and high-dose group) on the immobile time of the forced swimming test of the test mice, and ** represents P<0.01;

[0153] Figure 12 The influence of the Hypericum perforatum L. polysaccharide (Example 1, Example 3 and Comparative Example 1) on the immobile time of the forced swimming test of the mice, and * represents P<0.05, and ** represents P<0.01.

[0154] Figure 13 The influence of the Hypericum perforatum L. polysaccharide on the body weight of the fatigue mice before and after administration, and * represents P<0.05, and ** represents P<0.01;

[0155] Figure 14 The influence of the Hypericum perforatum L. polysaccharide on the exhaustive swimming time of the fatigue mice before and after administration, and * represents P<0.01. DETAILED DESCRIPTION

[0156] The detailed content of the present application is further explained and described below in combination with specific embodiments, but the protection scope of the present application is not limited thereby.

[0157] Example 1 Preparation of Hypericum perforatum polysaccharide

[0158] The preparation of Hypericum perforatum polysaccharide comprises the following steps:

[0159] (1) Take 200g of Hypericum perforatum, add water according to a material-to-liquid ratio of 1:10 (m / v, g / mL), soak for 1h, heat to 100°C and extract for 1h, filter, and collect the filtrate and the residue;

[0160] (2) In the residue collected in step (1), add water according to a material-to-liquid ratio of 1:10 (m / v, g / mL), heat to 100°C and extract for 1h, filter, and collect the filtrate and the residue;

[0161] (3) In the residue collected in step (2), add water according to a material-to-liquid ratio of 1:10 (m / v, g / mL), heat to 100°C and extract for 1h, filter, and collect the filtrate and the residue;

[0162] (4) Centrifuge (3000rpm*10min) the filtrate collected in the three times, collect the supernatant, and concentrate (60°C) under reduced pressure to a crude drug concentration of 1 (m / v, g / mL), add 95% ethanol according to a volume ratio of the concentrated solution:95% ethanol of 1:3, stand for 48h, centrifuge (3000rpm*10min), and obtain the supernatant and the precipitate;

[0163] (5) Add 1000mL of water to the precipitate prepared in step (4), stir and dissolve (1000rpm*30min), centrifuge (3000rpm*10min), and collect the supernatant and the precipitate;

[0164] (6) Add 1000mL of water to the precipitate collected in step (5), stir and dissolve (1000rpm*30min), centrifuge (3000rpm*10min), and collect the supernatant and the precipitate;

[0165] (7) Add 1000mL of water to the precipitate collected in step (6), stir and dissolve (1000rpm*30min), centrifuge (3000rpm*10min), and collect the supernatant and the precipitate;

[0166] (8) Combine the supernatants collected in steps (5)-(7), concentrate (60°C) under reduced pressure to a crude drug concentration of 1 (m / v, g / mL), and freeze-dry to obtain 10.1g of Hypericum perforatum polysaccharide.

[0167] Example 2 Preparation of Hypericum perforatum polysaccharide

[0168] The preparation of Hypericum perforatum polysaccharide comprises the following steps:

[0169] (1) Take 200 g of Hypericum perforatum, add 70% ethanol at a material-liquid ratio of 1:10 (m / v, g / mL), heat to 65°C, and reflux extract for 1 h. Filter and collect the residue;

[0170] (2) In the residue of step (1), add 70% ethanol at a material-liquid ratio of 1:10 (m / v, g / mL), heat to 65°C, and reflux extract for 1 h. Filter and collect the residue;

[0171] (3) Combine the residues of steps (1)-(2), add 2000 mL of water, heat to 100°C, and extract for 1 h. Filter and collect the filtrate and residue;

[0172] (4) In the residue of step (3), add 2000 mL of water, heat to 100°C, and extract for 1 h. Filter and collect the filtrate and residue;

[0173] (5) In the residue of step (4), add 2000 mL of water, heat to 100°C, and extract for 1 h. Filter and collect the filtrate;

[0174] (6) Combine the filtrates collected in steps (3)-(5), centrifuge (3000 rpm*10 min), collect the supernatant, and concentrate (60°C) under reduced pressure to a crude drug concentration of 1 (m / v, g / mL). Add 95% ethanol to the concentrated solution at a volume ratio of 1:3, stand for 48 h, centrifuge (3000 rpm*10 min), and obtain the supernatant (the ethanol volume concentration of the supernatant is about 75%) and the precipitate;

[0175] (7) Add 1000 mL of water to the precipitate prepared in step (6), stir to dissolve (1000 rpm*30 min), centrifuge (3000 rpm*10 min), and collect the supernatant and the precipitate;

[0176] (8) Add 1000 mL of water to the precipitate collected in step (7), stir to dissolve (1000 rpm*30 min), centrifuge (3000 rpm*10 min), and collect the supernatant and the precipitate;

[0177] (9) Add 1000 mL of water to the precipitate collected in step (8), stir to dissolve (1000 rpm*30 min), centrifuge (3000 rpm*10 min), and collect the supernatant and the precipitate;

[0178] (10) Combine the supernatants collected in steps (7)-(9), concentrate (60°C) under reduced pressure to a crude drug concentration of 1 (m / v, g / mL), and freeze-dry to obtain 6.2 g of Hypericum perforatum polysaccharide.

[0179] Example 3Preparation of Hypericum perforatum L. polysaccharide

[0180] The preparation of Hypericum perforatum L. polysaccharide comprises the following steps:

[0181] (1) Take 200 g of Hypericum perforatum L., and add 70% ethanol according to a material-liquid ratio of 1:10 (m / v, g / mL), heat to 65°C, reflux extraction for 1 h, filter, and collect the residue;

[0182] (2) In the residue of step (1), add 70% ethanol according to a material-liquid ratio of 1:10 (m / v, g / mL), heat to 65°C, reflux extraction for 1 h, filter, and collect the residue;

[0183] (3) Combine the residues of steps (1)-(2), add 2000 mL of water, heat to 100°C, extract for 1 h, filter, and collect the filtrate and residue;

[0184] (4) In the residue of step (3), add 2000 mL of water, heat to 100°C, extract for 1 h, filter, and collect the filtrate and residue;

[0185] (5) In the residue of step (4), add 2000 mL of water, heat to 100°C, extract for 1 h, filter, and collect the filtrate;

[0186] (6) Combine the filtrates collected in steps (3)-(5), centrifuge (3000 rpm*10 min), collect the supernatant, and concentrate (60°C) under reduced pressure to a crude drug concentration of 1 (m / v, g / mL); add 95% ethanol to the concentrated solution according to a volume ratio of 1:3, stand for 48 h, centrifuge (3000 rpm*10 min), and obtain the supernatant (the ethanol concentration of the supernatant is about 75%) and the precipitate;

[0187] (7) Add 1000 mL of water to the precipitate prepared in step (6), stir and dissolve (1000 rpm*30 min), centrifuge (3000 rpm*10 min), and collect the supernatant and the precipitate;

[0188] (8) Add 1000 mL of water to the precipitate collected in step (7), stir and dissolve (1000 rpm*30 min), centrifuge (3000 rpm*10 min), and collect the supernatant and the precipitate;

[0189] (9) Add 1000 mL of water to the precipitate collected in step (8), stir and dissolve (1000 rpm*30 min), centrifuge (3000 rpm*10 min), and collect the supernatant and the precipitate;

[0190] (10) Combine the supernatant collected in steps (7)-(9), concentrate (60℃) under reduced pressure to a crude drug concentration of 1 (m / v, g / mL), freeze-dry to obtain Hypericum perforatum polysaccharide 4.8 g.

[0191] Comparative Example 1 Preparation of Hypericum perforatum polysaccharide

[0192] The preparation of Hypericum perforatum polysaccharide comprises the following steps:

[0193] (1) Take 200 g of Hypericum perforatum, add water at a material-to-liquid ratio of 1:5 (m / v, g / mL), soak for 1 h, extract at 100℃ for 1 h, filter to obtain filtrate and residue;

[0194] (2) In the residue collected in step (1), add water at a material-to-liquid ratio of 1:5 (m / v, g / mL), soak for 1 h, extract at 100℃ for 1 h, filter to obtain filtrate and residue;

[0195] (3) Combine the filtrates of steps (1)-(2), filter while hot using activated carbon with a mass fraction of 0.5%, and centrifuge (3000 r / min x 10 min) to obtain supernatant;

[0196] Concentrate the obtained supernatant under reduced pressure to 200 mL, extract with 1-fold amount of petroleum ether, operate 3 times; separate the water phase, evaporate to extract residue, add ethanol to the water layer until the alcohol content reaches 80%, stand for 24 h, and then centrifuge (3000 r / min x 10 min) to obtain supernatant and precipitate;

[0197] (4) Add 100 mL of ethanol to the precipitate obtained in step (3), stir and centrifuge, collect the precipitate, operate 3 times; add 100 mL of acetone to the precipitate, stir and centrifuge, collect the precipitate, operate 3 times; dry at 80℃ to obtain Hypericum perforatum polysaccharide 6.3 g.

[0198] Test Example 1 Detection and structural characterization of Hypericum perforatum polysaccharide

[0199] 1. Detect the composition of Hypericum perforatum polysaccharide of Examples 1-3 according to the method of the present application, and the results are shown in Table 1.

[0200] Table 1

[0201]

[0202] 2. Detect the monosaccharide composition of Hypericum perforatum polysaccharide prepared in Examples 1-3 of the present application according to the method of the present application, and the results are shown in Table 2, Figure 1 , Figure 4 and Figure 7 .

[0203] Table 2

[0204] Number Monosaccharide composition of Hypericum perforatum polysaccharide Example 1 Molar ratio of arabinose:glucose:galactose:mannose:galacturonic acid in Hypericum perforatum polysaccharide was 1.00:9.03:2.44:0.75:2.32 Example 2 Molar ratio of arabinose:glucose: rhamnose:galactose:mannose in Hypericum perforatum polysaccharide was 1.00:2.57:0.25:1.52:0.36 Example 3 Molar ratio of arabinose:glucose:galactose:mannose:galacturonic acid in Hypericum perforatum polysaccharide was 1.00:1.42:1.41:0.19:0.57

[0205] 3. The molecular weight distribution of the Hypericum perforatum polysaccharide prepared in Example 1-3 according to the method of the present application was detected, and the results are shown in Figure 2 (A-chromatographic column TSKsw2500, B-chromatographic column TSKsw3000), Figure 5 (chromatographic column TSKsw3000), and Figure 8 (chromatographic column TSKsw3000).

[0206] 4. The infrared spectrum of the Hypericum perforatum polysaccharide prepared in Example 1-3 according to the method of the present application was detected, and the results are shown in Figure 3 , Figure 6 and Figure 9 .

[0207] Test Example 2 Study on the antidepressant effect of the Hypericum perforatum polysaccharide of the present application

[0208] Fifty male SPF level ICR mice with a body weight of 18-20 g (purchased from Sibeifu (Beijing) Experimental Animal Technology Co., Ltd.) were selected. After the test animals were adaptively fed for 1 week, they were randomly divided into 5 groups according to the body weight, 10 in each group.

[0209] Solvent group: intragastrically administered with the same volume of water;

[0210] Positive control group: Lu You Tai 150 mg / kg (commercially available, prepared with water);

[0211] Low-dose group: Hypericum perforatum polysaccharide of Example 3, 50 mg / kg;

[0212] Medium-dose group: Hypericum perforatum polysaccharide of Example 3, 200 mg / kg;

[0213] High-dose group: Hypericum perforatum polysaccharide of Example 3, 800 mg / kg.

[0214] The test animals were intragastrically administered once a day, and the intragastric administration was continuously performed for 8 days. On the 8th day, the forced swimming test was performed on the test animals 60 min after the intragastric administration. The mice were placed in a circular glass container with a height of 25 cm, a diameter of 10 cm, and a water depth of 19 cm, and the water temperature was 25℃. The cumulative immobile time in the last 4 min was recorded after 6 min of observation. The standard for determining immobility was that the animal stopped struggling in the water, became a floating state, and only had slight limb movement to keep the head floating on the water surface.

[0215] The experimental results are expressed as mean ± standard deviation. Data analysis was performed using GraphPad Prism 9.0.0, t-test or non-parametric test was used for comparison between two groups; one-way ANOVA (post-hoc test using Dunnett's method) or non-parametric test was used for comparison among multiple groups, and the difference was statistically significant (P<0.05).

[0216] (1) Record and compare the body weight of mice in each group before administration (day 0, see Figure 10 a) and after administration (day 8, see Figure 10 b).

[0217] Compared with the solvent group, there was no significant difference in the body weight of mice in each group before administration (P>0.1); compared with the solvent group, the body weight of mice in the positive control group was significantly lower (P=0.063); compared with the positive control group, the body weight of mice in the high-dose group was significantly higher (P=0.090).

[0218] (2) Compared with the solvent group, the positive control group can shorten the forced swimming immobility time of mice (P=0.082). The low-dose group, the medium-dose group and the high-dose group of Hypericum perforatum polysaccharide prepared in Example 3 all significantly shortened the forced swimming immobility time of mice (P<0.01) (see Figure 11 ).

[0219] Test Example 3 Antidepressant effect of Hypericum perforatum polysaccharide

[0220] Forty male SPF ICR mice weighing 18-20 g (purchased from Sibeifu (Beijing) Experimental Animal Technology Co., Ltd.) were selected. After adaptive feeding for 1 week, the test animals were randomly divided into 4 groups according to body weight, 10 in each group.

[0221] Solvent group: intragastrically administered with equal volume of water;

[0222] Model group: intragastrically administered with equal volume of water;

[0223] Positive control group: Lu You Tai 150 mg / kg (commercially available, prepared with water);

[0224] Test group: Hypericum perforatum polysaccharide 200 mg / kg of Example 3.

[0225] The test animals were intragastrically administered once a day. Continuous intragastric administration was performed for 9 days. On the 9th day, the test animals were subjected to reserpine antagonistic experiment 60 min after intragastric administration. The solvent group was intraperitoneally injected with 0.2% glacial acetic acid, and the model group, the positive control group and the test group were intraperitoneally injected with reserpine (2.5 mg / kg, dissolved in 0.2% glacial acetic acid), and the following indicators were observed and determined:

[0226] (1) Eyelid ptosis: After 2h of reserpine administration, the eyelid closure degree of mice was scored, 0 for complete opening, 1 / 4 closure for 1, 1 / 2 closure for 2, 3 / 4 closure for 3, and full closure for 4. The average score of each group was counted.

[0227] (2) Akinesia: After 2h of reserpine administration, the mice were placed in the center of a 7.5cm diameter circle for observation, and those unable to walk out of the circle within 15s were recorded as "akinesia". The number of akinesia mice in each group was recorded.

[0228] (3) Anus temperature: The anus temperature of mice was detected by electronic thermometer before administration (T0) and 3h after reserpine administration (T1). The difference value ΔT (ΔT = T0-T1) of two anus temperatures was counted.

[0229] The experimental results are expressed as mean ± standard deviation. Data analysis was performed using GraphPad Prism 9.0.0, t-test or non-parametric test was used for comparison between two groups; single factor analysis of variance (post-hoc test using Dunnett method) or non-parametric test was used for comparison among multiple groups. The difference was statistically significant (P<0.05). The results are shown in Table 3.

[0230] The model group mice showed obvious eyelid ptosis, body temperature drop, akinesia and other symptoms, and the modeling was successful (P<0.001). Compared with the model group, the positive control drug reversed the eyelid ptosis and akinesia of the test animals caused by reserpine after 9 days of continuous gavage (P<0.05); the test group significantly reversed the eyelid ptosis and akinesia of the test animals (P<0.05, P<0.01 or P<0.001), and the reversing effect was significantly better than that of the positive control drug Luoyoute.

[0231] Table 3

[0232]

[0233] Compared with the blank group, *** represents P<0.001; compared with the model group, # represents P<0.05, ## represents P<0.01, and ### represents P<0.001.

[0234] Test Example 4 Study on the antidepressant effect of Hypericum perforatum polysaccharide

[0235] SPF level, male ICR mice weighing 18-20g were selected, 50 in total (purchased from Sibeifu (Beijing) Experimental Animal Technology Co., Ltd.). After the mice were purchased, they were adaptively fed for 1 week, and then randomly divided into 5 groups according to body weight, 10 in each group.

[0236] Blank group: gavage with equal volume of water;

[0237] Positive control group: duloxetine 20 mg / kg;

[0238] Test group 1: Hypericum perforatum polysaccharide in Example 1 200 mg / kg;

[0239] Test group 2: Hypericum perforatum polysaccharide in Example 3 200 mg / kg;

[0240] Control group: Hypericum perforatum polysaccharide in Comparative Example 1 200 mg / kg.

[0241] The test animals were given a single gavage, and the forced swimming test was performed 60 min after administration. The mice were placed in a circular glass container with a height of 25 cm, a diameter of 10 cm, and a water depth of 19 cm, with water temperature at 25 ℃, and observed for 6 min, and the cumulative immobility time in the last 4 min was recorded. The criterion for immobility was that the animal stopped struggling in the water, became a floating state, and only had small limb movements to keep the head floating on the water surface.

[0242] The experimental results are expressed as mean ± standard deviation. Data analysis was performed using GraphPad Prism 9.0.0, and t-test or non-parametric test was used for comparison between two groups.

[0243] The results are shown in Figure 12 * indicates a significant difference (P < 0.05) compared with the blank group, ** indicates P < 0.01, and *** indicates P < 0.001.

[0244] Test Example 5 Study on the anti-fatigue effect of Hypericum perforatum polysaccharide

[0245] Forty male SPF ICR mice weighing 18-20 g (purchased from Sperof (Beijing) Experimental Animal Technology Co., Ltd.) were selected. After the mice were purchased, they were adaptively fed for 1 week, and then randomly divided into 4 groups according to body weight, 10 mice in each group.

[0246] Blank group: gavage with equal volume of water;

[0247] Model group: gavage with equal volume of water;

[0248] Positive control group: 10 mg / kg caffeine;

[0249] Test group: 200 mg / kg Hypericum perforatum polysaccharide in Example 3.

[0250] From the 1st to the 8th day, the mice in the blank group did not receive any training; the mice in the model group, the positive control group and the test group received training, in which they received 40 min of weight-loaded swimming training (the tail was tied with lead skin weighing 8% of the body weight, and the water surface height could prevent the mice from drowning) in the morning and 30 min of treadmill training (KW-PT small animal treadmill, Nanjing Calvin Biological Technology Co., Ltd.) in the afternoon.

[0251] From the 9th to the 15th day, the mice in the blank group were given intragastrically the same volume of water and did not receive any training; the mice in the model group, the positive control group and the test group were given intragastrically water, caffeine and the Hypericum perforatum polysaccharide obtained in Example 3 respectively, and received training, in which they received 40 min of weight-loaded swimming training (the tail was tied with lead skin weighing 8% of the body weight, and the water surface height could prevent the mice from drowning) in the morning and 30 min of treadmill training (KW-PT small animal treadmill, Nanjing Calvin Biological Technology Co., Ltd.) in the afternoon. On the 16th day, the exhaustive swimming experiment under weight was performed.

[0252] The experimental results are expressed as mean ± standard deviation. Data analysis was performed using GraphPad Prism 9.0.0, and t-test or non-parametric test was used for comparison between two groups. The difference was statistically significant (P<0.05). (1) The body weights of the mice in each group before administration (0th day, see Figure 13 a) and after administration (16th day, see Figure 13 b) were recorded and compared.

[0253] Compared with the blank group, 8 days of weight-loaded swimming training and treadmill training could cause the body weight of the mice in each group to decrease (P<0.05 or P<0.01). Compared with the model group, 8 days of intragastric administration of 200 mg / kg of the Hypericum perforatum polysaccharide of Example 3 could improve the body weight decrease of the model mice (P<0.05). Compared with the model group, there was no significant difference in the body weight of the mice in the positive control group (caffeine) (P>0.1).

[0254] (2) Exhaustive swimming experiment under weight: the exhaustive swimming experiment under weight was performed 60 min after administration on the 16th day. The tail of the mouse was tied with lead skin weighing 8% of the body weight, and the exhaustive swimming time before administration ( Figure 14 a) and the exhaustive swimming time after administration ( Figure 14 b) were counted.

[0255] The results showed that 8 days of weight-loaded swimming training and treadmill training could shorten the exhaustive swimming time under weight of the mice in each group (P=0.083 or P<0.05). Compared with the model group, the Hypericum perforatum polysaccharide of the application could increase the exhaustive swimming time under weight of the model mice (P<0.05), and its anti-fatigue effect was better than that of caffeine.

[0256] The above description of the embodiments is only used to help understand the method of the present application and its core idea. It should be noted that, for those skilled in the art, some improvements and modifications can be made to the present application without departing from the principles of the present application, and these improvements and modifications also fall within the protection scope of the claims of the present application.

Claims

1. A Hypericum perforatum L. polysaccharide having an anti-depression efficacy, wherein the weight average molecular weight (Mw) of the Hypericum perforatum L. polysaccharide is 200 Da-70000 Da, and the preparation of the Hypericum perforatum L. polysaccharide comprises the following steps: (1) adding water to Hypericum perforatum L. at a solid-liquid ratio of 1:1-15, soaking for 0.5-4 h, extracting at 90-100 ℃ for 0.5-5 h, filtering, and collecting the filtrate and the residue; (2) repeating the extraction of the residue according to the extraction method of step (1) for 1-3 times, and collecting the filtrate; (3) combining the collected filtrates of steps (1)-(2), centrifuging at a speed of 2000-4000 rpm for 5 min-30 min, concentrating the collected supernatant under reduced pressure to a crude drug concentration of 1:0.5-5, cooling to room temperature, adding 95% ethanol solution to the concentrated solution at a volume ratio of 1:1-5, standing for 24-72 h, centrifuging at a speed of 2000-4000 rpm for 5 min-30 min, and taking the supernatant and the precipitate; wherein the crude drug concentration refers to the mass volume ratio of the mass of crude drug to the mass of the concentrated solution; (4) adding water to the collected precipitate of step (3) at a mass volume ratio of 1:1-10, stirring and dissolving, stirring at a speed of 100-1000 rpm for 5 min-30 min, centrifuging at a speed of 2000-4000 rpm for 5 min-30 min, and collecting the supernatant and the precipitate; (5) repeating the purification of the collected precipitate of step (4) according to the purification method of step (4) for 1-4 times; (6) combining the collected supernatants of steps (4)-(5), and drying the supernatant under reduced pressure at 50-80 ℃ to obtain the Hypericum perforatum L. polysaccharide; Alternatively, the preparation of the Hypericum perforatum L. polysaccharide comprises the following steps: S-1: adding an ethanol solution with a concentration of 50-80% to Hypericum perforatum L. at a solid-liquid ratio of 1:1-15, soaking for 0.5-4 h, extracting at 65-100 ℃ by reflux for 0.5-5 h, filtering, and collecting the residue; S-2: repeating the extraction of the collected residue according to the extraction method of step S-1 for 1-2 times, filtering, and collecting the residue; S-3: adding water to the collected residue at a solid-liquid ratio of 1:1-15, extracting at 90-100 ℃ for 0.5-5 h, filtering, and collecting the filtrate and the residue; S-4: repeating the extraction of the residue according to the extraction method of step S-3 for 1-4 times, and collecting the filtrate; S-5: combining the collected filtrates of steps S-3 to S-4, centrifuging at a speed of 2000-4000 rpm for 5 min-30 min, concentrating the collected supernatant under reduced pressure to a crude drug concentration of 1:0.5-5, cooling to room temperature, adding 95% ethanol solution to the concentrated solution at a volume ratio of 1:1-5, standing for 24-72 h, centrifuging at a speed of 2000-4000 rpm for 5 min-30 min, and taking the supernatant and the precipitate. S-6: Add water to the collected precipitate in step S-5 according to the mass-volume ratio of crude medicinal material: water of 1:1-10, stir to dissolve, stirring speed 100-1000 rpm, stirring time 5 min-30 min, centrifugal speed 2000-4000 rpm, centrifugal time 5 min-30 min, collect supernatant and precipitate; S-7: Refer to the purification method of step S-6, the collected precipitate is repeatedly purified 1-4 times; S-8: Combine the supernatant collected in steps S-6 to S-7, and place it at 50-80℃ under reduced pressure to concentrate to a crude medicinal concentration of 0.5-2, and dry it to obtain the product.

2. The Hypericum perforatum polysaccharide of claim 1, wherein the weight average molecular weight (Mw) of the Hypericum perforatum polysaccharide is 250 Da-68000 Da.

3. The Hypericum perforatum polysaccharide of claim 1, wherein the Hypericum perforatum polysaccharide contains neutral sugar 20-80%, uronic acid 10-70% and protein 5-40% by weight percentage.

4. The Hypericum perforatum polysaccharide of claim 1, wherein the Hypericum perforatum polysaccharide comprises arabinose, glucose, galactose, mannose, galacturonic acid, rhamnose.

5. The Hypericum perforatum polysaccharide of claim 4, wherein the molar ratio of arabinose: glucose: galactose: mannose: galacturonic acid in the Hypericum perforatum polysaccharide is 1:0.1-10:0.1-10:0.1-10:0.1-10.

6. The Hypericum perforatum polysaccharide of claim 5, wherein the molar ratio of arabinose: glucose: galactose: mannose: galacturonic acid in the Hypericum perforatum polysaccharide is 1:1-10:1-5:0.1-1:0.1-5.

7. The Hypericum perforatum polysaccharide of claim 6, wherein the molar ratio of arabinose: glucose: galactose: mannose: galacturonic acid in the Hypericum perforatum polysaccharide is 1.00:1.42:1.41:0.19:0.

57.

8. The Hypericum perforatum polysaccharide of claim 6, wherein the molar ratio of arabinose: glucose: galactose: mannose: galacturonic acid in the Hypericum perforatum polysaccharide is 1.00:9.03:2.44:0.75:2.

32.

9. The Hypericum perforatum polysaccharide of claim 4, wherein the molar ratio of arabinose: glucose: rhamnose: galactose: mannose in the Hypericum perforatum polysaccharide is 1:0.1-10:0.1-10:0.1-10:0.1-10.

10. The Hypericum perforatum polysaccharide of claim 9, wherein the molar ratio of arabinose: glucose: rhamnose: galactose: mannose in the Hypericum perforatum polysaccharide is 1:1-5:0.1-1:1-5:0.1-1.

11. The Hypericum perforatum polysaccharide of claim 10, wherein the molar ratio of arabinose: glucose: rhamnose: galactose: mannose in the Hypericum perforatum polysaccharide is 1.00:2.57:0.25:1.52:0.

36.

12. The Hypericum perforatum polysaccharide of claim 1, wherein the ratio of material to liquid in step (1), S-1 or S-3 is 1:4-12.

13. The Hypericum perforatum polysaccharide of claim 12, wherein the ratio of material to liquid in step (1), S-1 or S-3 is 1:5-10.

14. The Hypericum perforatum polysaccharide of claim 1, wherein the soaking time in step (1) or S-1 is 1-3 h.

15. The Hypericum perforatum polysaccharide of claim 1, wherein the concentration of the ethanol solution in step S-1 is 60-70%.

16. The Hypericum perforatum polysaccharide of claim 1, wherein the extraction time in step (1), (2), S-1, S-2, S-3 or S-4 is 1-3 h.

17. The Hypericum perforatum polysaccharide of claim 1, wherein the number of times of repeated extraction of the residue in step (2) or S-4 is 2-3.

18. The Hypericum perforatum polysaccharide of claim 1, wherein the concentration of the crude drug in step (3), (6), S-5 or S-8 is 1-2.

19. The Hypericum perforatum polysaccharide of claim 1, wherein the volume ratio of the concentrated solution to 95% ethanol in step (3) or S-5 is 1:1-3.

20. The Hypericum perforatum polysaccharide of claim 1, wherein the mass-volume ratio of the crude drug to water in step (4) or S-6 is 1:5-8.

21. The Hypericum perforatum polysaccharide of claim 1, wherein the number of times of purification of the precipitate in step (5) or S-7 is 2-3.

22. The Hypericum perforatum polysaccharide of claim 1, wherein the drying in step (6) or S-8 is selected from any one or a combination of reduced pressure drying, spray drying and freeze drying.

23. The method for preparing the Hypericum perforatum polysaccharide having the efficacy of preventing and treating depression according to any one of claims 1-22, comprising the following steps: (1) adding water to Hypericum perforatum at a ratio of 1:1-15, soaking for 0.5-4 h, extracting at a temperature of 90-100 °C for 0.5-5 h, filtering, collecting the filtrate and residue; (2) repeating the extraction of the residue according to the extraction method of step (1) for 1-3 times, and collecting the filtrate; (3) combining the collected filtrates of steps (1)-(2), centrifuging at a speed of 2000-4000 rpm for 5 min-30 min, reducing the pressure to concentrate the collected supernatant to a crude drug concentration of 1:0.5-5, cooling to room temperature, adding 95% ethanol solution to the concentrated solution at a volume ratio of 1:1-5, standing for 24-72 h, centrifuging at a speed of 2000-4000 rpm for 5 min-30 min, and collecting the supernatant and precipitate; the crude drug concentration refers to the mass-volume ratio of crude drug to concentrated solution; (4) adding water to the collected precipitate of step (3) at a mass-volume ratio of 1:1-10, stirring to dissolve, stirring at a speed of 100-1000 rpm for 5 min-30 min, centrifuging at a speed of 2000-4000 rpm for 5 min-30 min, and collecting the supernatant and precipitate; (5) repeating the purification of the collected precipitate of step (4) according to the purification method of step (4) for 1-4 times; (6) drying the collected precipitate of step (5) by any one or a combination of reduced pressure drying, spray drying and freeze drying. (6) combine the supernatant collected in steps (4)-(5), and concentrate under reduced pressure at 50-80°C to a crude drug concentration of 0.5-2, and dry to obtain; Alternatively, the preparation of Hypericum perforatum polysaccharide comprises the following steps: S-1: add an ethanol solution with a concentration of 50-80% to Hypericum perforatum at a solid-liquid ratio of 1:1-15, soak for 0.5-4 h, warm to 65-100°C, and reflux extract for 0.5-5 h, filter, and collect the residue; S-2: according to the extraction method of step S-1, reflux extract the collected residue for 1-2 times, filter, and collect the residue; S-3: add water to the collected residue at a solid-liquid ratio of 1:1-15, warm to 90-100°C, extract for 0.5-5 h, filter, and collect the filtrate and residue; S-4: according to the extraction method of step S-3, repeat the extraction of the residue for 1-4 times, and collect the filtrate; S-5: combine the filtrate collected in steps S-3 to S-4, centrifuge at 2000-4000 rpm for 5 min-30 min, concentrate the collected supernatant under reduced pressure to a crude drug concentration of 1:0.5-5, cool to room temperature, add a 95% ethanol solution to the concentrated solution at a volume ratio of 1:1-5, stand for 24-72 h, centrifuge at 2000-4000 rpm for 5 min-30 min, and take the supernatant and precipitate; S-6: according to a solid-liquid ratio of 1:1-10, add water to the precipitate collected in step S-5, stir to dissolve, stir at a speed of 100-1000 rpm for 5 min-30 min, centrifuge at 2000-4000 rpm for 5 min-30 min, and collect the supernatant and precipitate; S-7: according to the purification method of step S-6, repeat the purification of the collected precipitate for 1-4 times; S-8: combine the supernatant collected in steps S-6 to S-7, and concentrate under reduced pressure at 50-80°C to a crude drug concentration of 0.5-2, and dry to obtain.

24. The method of claim 23, wherein the solid-liquid ratio in step (1), S-1 or S-3 is 1:4-12.

25. The method of claim 24, wherein the solid-liquid ratio in step (1), S-1 or S-3 is 1:5-10.

26. The method of claim 23, wherein the soaking time in step (1) or S-1 is 1-3 h.

27. The method of claim 23, wherein the concentration of the ethanol solution in step S-1 is 60-70%.

28. The method of claim 23, wherein the extraction time in step (1), (2), S-1, S-2, S-3 or S-4 is 1-3 h.

29. The method of claim 23, wherein the residue is repeatedly extracted for 2-3 times in step (2) or S-4.

30. The method of claim 29, wherein the crude drug concentration in step (3), (6), S-5 or S-8 is 1-2.

31. The method of claim 23, the concentration solution in step (3) or S-5: the volume ratio of 95% ethanol is 1:1-3.

32. The method of claim 31, the crude medicinal material in step (4) or S-6: the mass volume ratio of crude medicinal material: water is 1:5-8.

33. The method of claim 23, the purification times of the precipitate in step (5) or S-7: 2-3 times.

34. The method of claim 23, the drying in step (6) or S-8: selected from one or a combination of reduced pressure drying, spray drying, freeze drying.

35. A Hypericum perforatum L. polysaccharide composition having the efficacy of preventing and treating depression, the composition consisting of the Hypericum perforatum L. polysaccharide of any one of claims 1-22 and a pharmaceutically acceptable carrier.

36. The composition of claim 35, the dosage form of the composition is oral preparation.

37. The composition of claim 36, the oral preparation is selected from one of tablet, capsule, granule, paste, syrup, powder, effervescent, suspension, pill, mixture.

38. The composition of claim 36, the pharmaceutically acceptable carrier comprises filler, disintegrant, binder, lubricant, dispersant, humectant, pH adjuster, osmotic pressure adjuster, pore-forming agent, solubilizer, suspending agent, emulsifier, co-emulsifier, lyophilization protective agent, flavoring agent.

39. Use of the Hypericum perforatum L. polysaccharide of any one of claims 1-22 or the Hypericum perforatum L. polysaccharide composition of any one of claims 35-38 in the preparation of a product for preventing and treating depression.

40. Use of the Hypericum perforatum L. polysaccharide of any one of claims 1-22 or the Hypericum perforatum L. polysaccharide composition of any one of claims 35-38 in the preparation of a product for anti-fatigue.

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