Inula flower extract as well as preparation method and application thereof

Abstract

The invention relates to an inula flower composition which is prepared by the following steps: (1) adding water into an inula flower extract to be purified according to the weight-volume ratio of the inula flower extract to the water being 1: (10-20), and performing ultrasonic dispersion to prepare a suspension; (2) adding petroleum ether into the prepared suspension according to the volume ratio of the petroleum ether to the suspension being (1-3): 1, performing ultrasonic extraction, removing a petroleum ether layer, and retaining a water layer; (3) repeating the ultrasonic extraction operation in the step (2) for 1-3 times, adding water to dilute a water layer, performing ultrasonic dispersion, filtering and collecting filtrate; (4) loading the filtrate on macroporous resin, carrying out static adsorption for 10-18 hours, collecting the effluent, and circularly loading the sample; and (5) sequentially eluting with deionized water, a 10% ethanol solution, a 20% ethanol solution, a 30% ethanol solution and a 50% ethanol solution, collecting the eluent eluted with the 30% ethanol solution and the eluent eluted with the 50% ethanol solution, concentrating, and drying to obtain the traditional Chinese medicine composition. The inula flower composition disclosed by the invention has excellent activity of resisting post-traumatic stress disorder (PTSD) and the like.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to an extract of Inula japonica, its preparation method, and its application. Background Technology

[0002] Inula japonica or Inula britannica are the dried flower heads of plants belonging to the genus Inula in the family Asteraceae. In my country, they are mainly distributed in Henan, Hebei, Jiangsu, Zhejiang, Xinjiang, and Northeast China. They are bitter, pungent, and salty in taste, and slightly warm in nature. Traditional Chinese medicine believes they have the functions of lowering qi, resolving phlegm, promoting diuresis, and stopping vomiting. They are often used to treat symptoms such as cough due to wind-cold, phlegm accumulation, chest tightness, wheezing with excessive phlegm, vomiting, belching, and epigastric fullness.

[0003] Modern research shows that Inula japonica extract has a variety of pharmacological activities, including antioxidant, antitumor, anti-inflammatory, antifungal, hypoglycemic, anti-constipation, anti-aging, and hypotensive effects. These effects are closely related to its complex chemical composition. Its main active ingredients include: (1) flavonoids, which are natural antioxidants and have antioxidant, anti-inflammatory, expectorant, antitussive, and cardiovascular protective effects; (2) terpenoids, which are the most important bioactive substances and exhibit significant antitumor, antibacterial, and anti-inflammatory activities; (3) volatile oils, which help with antibacterial, expectorant, and antiasthmatic effects and can relieve respiratory symptoms; (4) organic acids, which have antibacterial, antiviral, antioxidant, and hepatoprotective and choleretic activities; and (5) sterols, which have anti-inflammatory, antitussive, and cholesterol-lowering effects. In addition, Inula japonica also contains polysaccharides (with immunomodulatory effects), amino acids, and inorganic elements.

[0004] In terms of extraction processes, common methods include water extraction and alcohol extraction. Alcohol extraction mainly yields fat-soluble components such as flavonoids, terpene lactones, and alkaloids, while water extraction can simultaneously extract water-soluble components such as polysaccharides, glycosides, amino acids, tannins, and organic acid salts, as well as some fat-soluble components. The different extraction methods directly lead to significant differences in the chemical composition of the extracts, thus affecting their pharmacological activity and final applications.

[0005] Post-traumatic stress disorder (PTSD), a major mental disorder triggered by traumatic experiences such as war, disasters, and violent events, has a wide-ranging and profound impact. It not only impairs mental health but also leads to physical health problems (such as chronic fatigue, increased risk of immune and cardiovascular diseases), decreased social functioning, and high-risk behaviors. Notably, PTSD was included in the "Classification and Catalogue of Occupational Diseases" in December 2024, recognizing it as an occupational disease for groups such as emergency responders. PTSD can be classified into acute (within 3 months), chronic (more than 3 months), and delayed-onset (onset after 6 months). Treatment methods include medication (such as serotonin reuptake inhibitors), psychotherapy, and physical therapy. However, the disease is prone to recurrence and relapse; approximately one-third of patients may develop chronic PTSD, which is lifelong, indicating that current treatments still have limitations.

[0006] Given the growing societal demand for PTSD treatment, there remains a pressing need to develop safer, more effective, and easier-to-use drug regimens. Inula japonica, with its multi-component and multi-target action characteristics, holds promise as a new candidate substance for the development of related functional products. Summary of the Invention

[0007] The purpose of this invention is to provide an Inula japonica composition, the preparation of which includes the following steps:

[0008] (1) Add water to the extract to be purified according to the weight-volume ratio of Inula japonica extract to water of 1:10-20, and disperse by ultrasonication to obtain a suspension.

[0009] (2) Add petroleum ether to the prepared suspension at a volume ratio of 1-3:1, perform ultrasonic extraction, remove the petroleum ether layer, and retain the aqueous layer.

[0010] (3) Repeat the ultrasonic extraction operation in step (2) 1-3 times, dilute the aqueous layer with water, disperse by ultrasonication, filter, and collect the filtrate;

[0011] (4) Load the filtrate onto macroporous resin and allow it to stand for 10-18 hours to adsorb. Collect the effluent and recycle it for further loading.

[0012] (5) Elute sequentially with deionized water, 10% ethanol solution, 20% ethanol solution, 30% ethanol solution and 50% ethanol solution. Collect the eluent eluted with 30% ethanol solution and the eluent eluted with 50% ethanol solution, concentrate and dry to obtain the final product.

[0013] In a preferred embodiment of the present invention, the ultrasonic conditions are 30-40 minutes of ultrasound at 40-50°C and an ultrasonic frequency of 30-40 kHz.

[0014] In the preferred embodiment of the present invention, in step (3), water is added to dilute the product by 1-3 times.

[0015] In the preferred embodiment of the present invention, in step (4), the macroporous adsorption resin is AB-8 resin.

[0016] In a preferred embodiment of the present invention, in step (4), the effluent is circulated and sampled 5-10 times.

[0017] In the preferred embodiment of the present invention, in step (4), the flow rate of the filtrate through the resin is 1-3 column volumes / h.

[0018] In the preferred embodiment of the present invention, in step (5), the amount of deionized water, 10% ethanol solution, 20% ethanol solution, 30% ethanol solution and 50% ethanol solution added is 2-5 times the column volume, preferably 3-4 times the column volume.

[0019] In a preferred embodiment of the present invention, in step (5), the volume ratio of the concentrated liquid to the volume weight of the Inula japonica extract to be purified is 1-2:1.

[0020] In the preferred embodiment of the present invention, in step (5), the concentration is selected from any one or a combination of rotary evaporation concentration, vacuum concentration, thin film concentration, membrane separation concentration, ultrafiltration, and nanofiltration.

[0021] In the preferred embodiment of the present invention, the concentration temperature in step (5) is 40℃-80℃, preferably 50℃-70℃.

[0022] In a preferred embodiment of the present invention, the drying process is selected from any one or a combination of spray drying, freeze drying, vacuum drying, and oven drying.

[0023] In a preferred embodiment of the present invention, the drying temperature is 40℃-80℃, preferably 50℃-70℃.

[0024] In a preferred embodiment of the present invention, the preparation of the Inula japonica extract includes the following steps:

[0025] (S-1) Add water to Inula japonica at a material-to-liquid ratio of 1:5–1:20 (g / mL), soak for 0.5–4 h, then heat to 30–100℃ and extract for 0.5–5 h. Filter and collect the filtrate and residue separately.

[0026] (S-2) Repeat the extraction of the filter residue obtained in step (S-1) 1–3 times according to the method in step (S-1), and combine the filtrates obtained from each extraction;

[0027] (S-3) Combine the filtrates from steps (S-1) and (S-2), centrifuge at 2000–4000 rpm for 5–30 min, collect the supernatant, concentrate under reduced pressure to a crude drug concentration of 1:0.5–1:5 (g / mL), and dry to obtain the final product.

[0028] In the preferred embodiment of the present invention, the material-liquid ratio in step (S-1) is 1:8-15, preferably 1:12-20.

[0029] In the preferred embodiment of the present invention, the extraction temperature in step (S-1) is 40-80℃, preferably 50-60℃.

[0030] In the preferred embodiment of the present invention, the extraction time in step (S-1) is 1-4 hours, preferably 2-4 hours.

[0031] In a preferred embodiment of the present invention, the soaking time in step (S-1) is 1-4 hours.

[0032] In the preferred embodiment of the present invention, the filter residue extraction in step (S-2) is performed 2-3 times.

[0033] In a preferred embodiment of the present invention, the concentration of the crude drug in step (S-3) is 1-2 (m / v).

[0034] In a preferred embodiment of the present invention, the drying in step (S-3) is selected from any one or a combination of vacuum drying, spray drying, and freeze drying.

[0035] Another object of the present invention is to provide a method for preparing an Inula japonica composition, comprising the following steps:

[0036] (1) Add water to the extract to be purified according to the weight-volume ratio of Inula japonica extract to water of 1:10-20, and disperse by ultrasonication to obtain a suspension.

[0037] (2) Add petroleum ether to the prepared suspension at a volume ratio of 1-3:1, perform ultrasonic extraction, remove the petroleum ether layer, and retain the aqueous layer.

[0038] (3) Repeat the ultrasonic extraction operation in step (2) 1-3 times, dilute the aqueous layer with water, disperse by ultrasonication, filter, and collect the filtrate;

[0039] (4) Load the filtrate onto macroporous resin and allow it to stand for 10-18 hours to adsorb. Collect the effluent and recycle it for further loading.

[0040] (5) The mixture was eluted sequentially with deionized water, 10% ethanol solution, 20% ethanol solution, 30% ethanol solution and 50% ethanol solution. The eluent eluted with 30% ethanol solution and the eluent eluted with 50% ethanol solution were collected, concentrated and dried to obtain the Inula japonica composition.

[0041] In a preferred embodiment of the present invention, the ultrasonic conditions are 30-40 minutes of ultrasound at 40-50°C and an ultrasonic frequency of 30-40 kHz.

[0042] In the preferred embodiment of the present invention, in step (3), water is added to dilute the product by 1-3 times.

[0043] In the preferred embodiment of the present invention, in step (4), the macroporous adsorption resin is AB-8 resin.

[0044] In the preferred embodiment of the present invention, in step (4), the effluent is circulated and sampled 5-10 times.

[0045] In the preferred embodiment of the present invention, in step (4), the flow rate of the filtrate through the resin is 1-3 column volumes / h.

[0046] In the preferred embodiment of the present invention, the amounts of deionized water, 10% ethanol solution, 20% ethanol solution, 30% ethanol solution and 50% ethanol solution added in step (5) are 1-5 times the column volume, preferably 3-4 times the column volume.

[0047] In a preferred embodiment of the present invention, the volume ratio of the concentrated liquid to the volume-to-weight ratio of the Inula japonica extract to be purified is 1-2:1.

[0048] In a preferred embodiment of the present invention, the concentration is selected from any one or a combination of rotary evaporation concentration, vacuum concentration, thin film concentration, membrane separation concentration, ultrafiltration, and nanofiltration.

[0049] In a preferred embodiment of the present invention, the concentration temperature is 40℃-80℃, preferably 50℃-70℃.

[0050] In a preferred embodiment of the present invention, the drying process is selected from any one or a combination of spray drying, freeze drying, and vacuum drying.

[0051] In a preferred embodiment of the present invention, the drying temperature is 40℃-80℃, preferably 50℃-70℃.

[0052] In a preferred embodiment of the present invention, the preparation of the Inula japonica extract includes the following steps:

[0053] (S-1) Add water to Inula japonica at a material-to-liquid ratio of 1:5–1:20 (g / mL), soak for 0.5–4 h, then heat to 30–100℃ and extract for 0.5–5 h. Filter and collect the filtrate and residue separately.

[0054] (S-2) Repeat the extraction of the filter residue obtained in step (S-1) 1–3 times according to the method in step (S-1), and combine the filtrates obtained from each extraction;

[0055] (S-3) Combine all the filtrates from steps (S-1) and (S-2), centrifuge at 2000–4000 rpm for 5–30 min, collect the supernatant, concentrate under reduced pressure to a crude drug concentration of 1:0.5–1:5 (g / mL), and dry to obtain the final product.

[0056] In the preferred embodiment of the present invention, the material-liquid ratio in step (S-1) is 1:8-15, preferably 1:12-20.

[0057] In the preferred embodiment of the present invention, the extraction temperature in step (S-1) is 40-80℃, preferably 50-60℃.

[0058] In the preferred embodiment of the present invention, the extraction time in step (S-1) is 1-4 hours, preferably 2-4 hours.

[0059] In a preferred embodiment of the present invention, the soaking time in step (S-1) is 1-4 hours.

[0060] In the preferred embodiment of the present invention, the filter residue extraction in step (S-2) is performed 2-3 times.

[0061] In a preferred embodiment of the present invention, the concentration of the crude drug in step (S-3) is 1-2 (m / v).

[0062] In a preferred embodiment of the present invention, the drying in step (S-3) is selected from any one or a combination of vacuum drying, spray drying, and freeze drying.

[0063] Another object of the present invention is to provide an Inula japonica extract, the preparation of which includes the following steps:

[0064] (S-1) Add water to Inula japonica at a material-to-liquid ratio of 1:5–1:20 (g / mL), soak for 0.5–4 h, then heat to 30–100℃ and extract for 0.5–5 h. Filter and collect the filtrate and residue separately.

[0065] (S-2) Repeat the extraction of the filter residue obtained in step (S-1) 1–3 times using the same method, and combine the filtrates obtained from each extraction;

[0066] (S-3) Combine all the filtrates from steps (S-1) and (S-2), centrifuge at 2000–4000 rpm for 5–30 min, collect the supernatant, concentrate under reduced pressure to a crude drug concentration of 1:0.5–1:5 (g / mL), and dry to obtain Inula japonica extract.

[0067] In the preferred embodiment of the present invention, the material-liquid ratio in step (S-1) is 1:8-15, preferably 1:12-20.

[0068] In the preferred embodiment of the present invention, the extraction temperature in step (S-1) is 40-80℃, preferably 50-60℃.

[0069] In the preferred embodiment of the present invention, the extraction time in step (S-1) is 1-4 hours, preferably 2-4 hours.

[0070] In a preferred embodiment of the present invention, the soaking time in step (S-1) is 1-4 hours.

[0071] In the preferred embodiment of the present invention, the filter residue extraction in step (S-2) is performed 2-3 times.

[0072] In a preferred embodiment of the present invention, the concentration of the crude drug in step (S-3) is 1-2 (m / v).

[0073] In a preferred embodiment of the present invention, the drying in step (S-3) is selected from any one or a combination of vacuum drying, spray drying, and freeze drying.

[0074] Another object of the present invention is to provide an Inula japonica pharmaceutical composition comprising any one or a combination of the Inula japonica extract of the present invention, the Inula japonica composition of the present invention, and a pharmaceutically acceptable carrier.

[0075] In a preferred embodiment of the present invention, the dosage form of the pharmaceutical composition is selected from either an oral preparation or a topical preparation.

[0076] In a preferred embodiment of the present invention, the oral preparation is selected from any one of oral liquid preparations, tablets, capsules, granules, syrups, powders, elixirs, effervescent tablets, suspensions, pills, drop pills, mixtures, pastes, emulsions, and liniments.

[0077] In a preferred embodiment of the present invention, the external preparation is selected from any one of gels, ointments, plasters, creams, ointments, liniments, lotions, suppositories, smears, gelling agents, ointments, aerosols, dry powder inhalers, sprays, nebulizers, and hydrogel patches.

[0078] In a preferred embodiment of the present invention, the injectable is selected from any one of solution-type injection, emulsion-type injection, suspension-type injection, sterile powder for injection, and large-volume parenteral solutions.

[0079] In a preferred embodiment of the present invention, the pharmaceutically acceptable carrier is a commonly used excipient or additive in the art for preparing the desired formulation, selected from any one or a combination of fillers (diluents), disintegrants, binders, lubricants (flow aids or anti-adhesion agents), dispersants, wetting agents, pH adjusters (acid-base adjusters), osmotic pressure adjusters, pore-forming agents, solubilizers, antioxidants, antibacterial agents (bactericides), analgesics (anesthetics), suspending agents, emulsifiers, co-emulsifiers, lyophilization protectants, flavoring agents, and fragrances.

[0080] In a preferred embodiment of the present invention, the filler is selected from any one or a combination of lactose, powdered sugar, dextrin, starch, sodium carboxymethyl starch, sodium starch glycolate, pregelatinized starch, modified starch, hydroxypropyl starch, potato starch, corn starch, cellulose, inorganic calcium salts, calcium chloride, calcium sulfate, calcium phosphate, calcium hydrogen phosphate, precipitated calcium carbonate, sorbitol, mannitol, microcrystalline cellulose, sodium carboxymethyl cellulose, ethyl cellulose, and hydroxypropyl methyl cellulose.

[0081] In a preferred embodiment of the present invention, the disintegrant is selected from any one or a combination of starch, sodium carboxymethyl starch, sodium starch glycolate, pregelatinized starch, modified starch, hydroxypropyl starch, potato starch, corn starch or its derivatives, croscarmellose, microcrystalline cellulose, low-substituted hydroxypropyl cellulose, and croscarmellose sodium.

[0082] In a preferred embodiment of the present invention, the adhesive is selected from any one or a combination of syrup, gum arabic, gelatin, sorbitol, astragalus gum, cellulose, microcrystalline cellulose, sodium carboxymethyl cellulose, ethyl cellulose, methyl cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, gelatin paste, starch paste, polyvinylpyrrolidone, sodium carboxymethyl starch, sodium starch glycolate, pregelatinized starch, modified starch, hydroxypropyl starch, potato starch, and corn starch.

[0083] In a preferred embodiment of the present invention, the lubricant is selected from any one or a combination of micronized silica gel, magnesium stearate, talc, colloidal silica, aluminum hydroxide, boric acid, hydrogenated vegetable oil, polyethylene glycol, and sodium dodecyl sulfate.

[0084] In a preferred embodiment of the present invention, the wetting agent is selected from any one or a combination of sodium dodecyl sulfate, polysorbate (Tween), water, alcohol, and ester.

[0085] In a preferred embodiment of the present invention, the antioxidant is selected from any one or a combination of sodium sulfite, sodium bisulfite, sodium metabisulfite, sodium thiosulfate, vitamin C, vitamin E, butylbenzoic acid, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), propyl gallate (PG), nicotinamide, acetylcysteine, and tert-butylhydroquinone (TBHQ).

[0086] In a preferred embodiment of the present invention, the antibacterial agent (bactericide) is selected from any one or a combination of phenol, cresol, chlorobutanol, and benzyl alcohol.

[0087] In a preferred embodiment of the present invention, the analgesic (anesthetic) is selected from any one or a combination of chlorobutanol, benzyl alcohol, lidocaine, and procaine.

[0088] In a preferred embodiment of the present invention, the suspending agent is selected from any one or a combination of microcrystalline cellulose, sodium carboxymethyl cellulose, ethyl cellulose, methyl cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, syrup, glycerin, gelatin, gum arabic, tragacanth gum, sodium alginate, potassium alginate, and pectin.

[0089] In a preferred embodiment of the present invention, the acid-base adjuster (pH adjuster) is selected from any one or a combination of hydrochloric acid, sodium hydroxide, potassium hydroxide, citric acid, sodium citrate, potassium citrate, malic acid, sodium malate, potassium malate, sodium dihydrogen phosphate, disodium hydrogen phosphate, calcium hydroxide, calcium lactate, sodium lactate, sodium phosphate, potassium phosphate, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, sodium bicarbonate, and sodium carbonate.

[0090] In a preferred embodiment of the present invention, the osmotic pressure regulator is selected from any one of sodium chloride, potassium chloride, glucose, phosphate, and citrate.

[0091] In a preferred embodiment of the present invention, the emulsifier is selected from any one or a combination of sodium stearate, potassium stearate, triethanolamine stearate, magnesium stearate, calcium stearate, sodium dodecyl sulfate, sodium dodecylbenzene sulfonate, polysorbate (Tween), Span, Mize, benzyl sulfadiazine, polyvinyl alcohol, tragacanth gum, gum arabic, Pronic F-68, lecithin, and soybean lecithin.

[0092] In a preferred embodiment of the present invention, the co-emulsifier is selected from any one or a combination of n-butanol, ethylene glycol, ethanol, propylene glycol, glycerol, and polyglycerol ester.

[0093] In a preferred embodiment of the present invention, the freeze-drying protectant is selected from any one or a combination of sucrose, lactose, galactose, glucose, trehalose, mannitol, sorbitol, cyclodextrin, and hydroxyethyl starch.

[0094] In a preferred embodiment of the present invention, the solubilizer is selected from any one or a combination of Tween-80, Prönnick F-68, Mize, Benzyl, cholate, deoxycholate, glycerol, propylene glycol, polyethylene glycol, hydroxypropyl-β-cyclodextrin, lauryl glucoside, cocoyl glucoside, and Span.

[0095] In a preferred embodiment of the present invention, the flavoring agent is selected from any one or a combination of honey, syrup, acid, alkali, flavoring, sweetener, acesulfame potassium, and aspartame.

[0096] In a preferred embodiment of the present invention, any one or a combination of the first extract and the second extract of Inula japonica of the present invention, together with a pharmaceutically acceptable sustained-release or controlled-release carrier, is prepared into a sustained-release or controlled-release formulation according to a method for preparing sustained-release or controlled-release formulations in the art.

[0097] In a preferred embodiment of the present invention, the sustained-release formulation carrier or controlled-release formulation carrier is selected from any one or a combination of oily incorporators, hydrophilic colloids, water-insoluble blockers, enteric blockers, and biodegradable blockers.

[0098] In a preferred embodiment of the present invention, the oily admixture is selected from any one or a combination of glyceryl monostearate, hydrogenated castor oil, mineral oil, polysiloxane, and dimethylsiloxane.

[0099] In a preferred embodiment of the present invention, the hydrophilic colloid is selected from any one or a combination of methylcellulose (MC), sodium carboxymethylcellulose (CMC-Na), hydroxyethylcellulose (HEC), hydroxypropylcellulose (HPC), hydroxypropyl methylcellulose (HMPC), polyvinylpyrrolidone (PVP), gum arabic, tragacanth gum or carboplatin, polyvinyl alcohol (PVA), pectin, alginate, chitosan, xanthan gum, guar gum, carrageenan, gelatin, agar, and galactomannan.

[0100] In a preferred embodiment of the present invention, the water-insoluble inhibitor is selected from any one or a combination of ethyl cellulose (EC), cellulose acetate (CA), polyethylene, polypropylene, polysiloxane, ethylene vinyl acetate copolymer (EVA), and polymethyl methacrylate.

[0101] In a preferred embodiment of the present invention, the enteric inhibitor is selected from any one or a combination of cellulose acetate acetate (CAP), hydroxypropyl methylcellulose phthalate (HMPCP), polyvinyl alcohol phthalate (PVAP), hydroxypropyl methylcellulose acetate succinate (HPMCAS), and acrylic resins.

[0102] In a preferred embodiment of the present invention, the biodegradable inhibitor is selected from any one or a combination of waxes, fatty acids, fatty acid esters, fatty alcohols, carnauba wax, stearic acid, glyceryl monostearate, stearyl alcohol, and cetyl alcohol.

[0103] In a preferred embodiment of the present invention, the administration method of the pharmaceutical composition is selected from any one of oral administration, mucosal administration, and skin administration.

[0104] Another object of the present invention is to provide the use of any one or a combination of the Inula japonica extract, Inula japonica composition, Inula japonica pharmaceutical composition of the present invention in the preparation of anti-stress products.

[0105] In a preferred embodiment of the present invention, the anti-stress product includes any one of the following: antidepressant products, anti-anxiety products, anti-post-traumatic stress disorder products, anti-fatigue products, and memory-improving products.

[0106] In a preferred embodiment of the present invention, the product is selected from any one of pharmaceuticals, food, functional food, health food, and special medical food.

[0107] Unless otherwise stated, the following terms are defined in this invention:

[0108] 1. The material-to-liquid ratio of the present invention is the mass-to-volume ratio (m / v) of the Inula japonica to be extracted and the extraction solvent (water).

[0109] 2. In this invention, the concentration of raw medicinal materials (also known as the amount of raw medicinal materials) refers to the amount of raw medicinal materials contained in a unit volume of preparation or extract.

[0110] Unless otherwise stated, when this invention relates to percentages between liquids, the percentage is volume / volume percentage; when this invention relates to percentages between liquids and solids, the percentage is volume / weight percentage; when this invention relates to percentages between solids and liquids, the percentage is weight / volume percentage; the remainder is weight / weight percentage.

[0111] Compared with the prior art, the present invention has the following beneficial technical effects:

[0112] 1. The Inula japonica extract, Inula japonica composition and pharmaceutical composition of the present invention have excellent anti-stress, anti-depressant, anti-fatigue, anti-PTSD, sleep improvement and memory improvement effects, and have the advantages of high bioavailability, safety and effectiveness.

[0113] 2. The preparation method of the present invention has the advantages of simple operation, high yield, better cost, green and environmentally friendly, and suitable for industrial production. Attached Figure Description

[0114] Figure 1 Example 1: HPLC characteristic chromatogram of Inula japonica extract;

[0115] Figure 2 Example 2: HPLC characteristic chromatogram of Inula japonica extract;

[0116] Figure 3 Example 3: HPLC characteristic chromatogram of Inula japonica extract;

[0117] Figure 4 Study on the anti-PTSD-like effect of the Inula japonica extract of the present invention;

[0118] Figure 5 Study on the anti-PTSD-like effect of the Inula japonica composition of the present invention. Detailed Implementation

[0119] The following detailed explanation and description of the present invention with reference to specific embodiments is not intended to limit the scope of protection of the present invention.

[0120] Example 1 Preparation of Inula japonica extract according to the present invention

[0121] The preparation of Inula japonica extract includes the following steps:

[0122] (1) Add 200g of Inula japonica to 2400mL of deionized water, soak for 30min, extract at room temperature for 4h, filter, and collect the filtrate and residue;

[0123] (2) Add 2400 mL of deionized water to the residue, soak for 30 min, extract at room temperature for 4 h, filter, and collect the filtrate;

[0124] (3) Combine the two filtrates, centrifuge (3000 rpm * 15 min), concentrate the filtrate under reduced pressure at 50-55℃ to a crude drug concentration of 1:1 (m / v), and dry the concentrate at 60℃ to obtain 33.0 g of Inula japonica extract.

[0125] Example 2 Preparation of Inula japonica extract according to the present invention

[0126] The preparation of Inula japonica extract includes the following steps:

[0127] (1) Add 200g of Inula japonica to 2400mL of deionized water, soak for 30min, then extract in a water bath at 50-55℃ for 4h, filter, and collect the filtrate and residue;

[0128] (2) Add 2400 mL of deionized water to the residue from step (1), soak for 30 min, extract in a water bath at 50-55℃ for 4 h, filter, and collect the filtrate.

[0129] (3) Combine the two filtrates, centrifuge (3000 rpm * 15 min), concentrate the filtrate under reduced pressure at 50-55℃ to a crude drug concentration of 1:1 (m / v), and dry the concentrate at 60℃ to obtain 40.4 g of Inula japonica extract.

[0130] Example 3 Preparation of Inula japonica extract according to the present invention

[0131] The preparation of Inula japonica extract includes the following steps:

[0132] (1) Add 100g of Inula japonica to 1200mL of deionized water, soak for 30min, then reflux and decoct for 2h, filter, and collect the filtrate and dregs;

[0133] (2) Add 1200mL of deionized water to the dregs from step (1), soak for 30min, reflux and decoct for 2h, filter and collect the filtrate;

[0134] (3) Combine the two filtrates, centrifuge (3000 rpm * 15 min), concentrate the filtrate under reduced pressure at 50-55℃ to a crude drug concentration of 1:1 (m / v), and dry the concentrate at 60℃ to obtain 24.5g of Inula japonica extract.

[0135] Example 4 Preparation of the Inula japonica composition of the present invention

[0136] (1) Add water to 30g of the Inula japonica extract prepared in Example 2 according to the weight-volume ratio of Inula japonica extract to water of 1:10, and disperse by ultrasonication (ultrasounding at 50 ℃ for 30 min, ultrasonic frequency 40KHz) to obtain a suspension.

[0137] (2) Add petroleum ether to the suspension prepared in step (1) according to the volume ratio of petroleum ether to suspension of 1:1, perform ultrasonic extraction, remove the petroleum ether layer, and retain the aqueous layer.

[0138] (3) Repeat the ultrasonic extraction operation in step (2) 3 times (i.e., add petroleum ether to the water layer in step (2) according to the volume ratio of petroleum ether to water layer of 1:1, remove the petroleum ether layer and keep the water layer), dilute the water layer with water to 900 mL, sonicate, filter, and collect the filtrate.

[0139] (4) Load the filtrate onto an AB-8 macroporous resin adsorption column at a flow rate of 3 BV / h, allow it to stand for 12 hours, collect the effluent and circulate it 10 times.

[0140] (5) The solution was eluted sequentially with 3 column volumes of deionized water, 3 column volumes of 10% ethanol solution, 3 column volumes of 20% ethanol solution, 3 column volumes of 30% ethanol solution, and 3 column volumes of 50% ethanol solution. The eluent obtained with 30% ethanol solution and the eluent obtained with 50% ethanol solution were collected and concentrated by rotary evaporation at 50°C until the volume of the concentrated solution was 60 mL, thus obtaining 4.8 g of the Inula japonica composition.

[0141] Experimental Example 1 HPLC detection of Inula japonica extract in this invention

[0142] Preparation of test solutions: Weigh 25 mg of the Inula japonica extract from Example 1, Example 2, and Example 3 respectively, place them in 10 mL centrifuge tubes, add 5 mL of 20% methanol, sonicate at 50 °C for 15 min, filter through a 0.45 μm microporous membrane, and take the filtrate for HPLC detection.

[0143] Standards: Rutin, Taxodiacetic acid, and Quercetin were purchased from the China National Institutes for Food and Drug Control, Shanghai Yuanye Biotechnology Co., Ltd., and Shanghai Taoshu Biotechnology Co., Ltd., respectively.

[0144] SHIMADZU high-performance liquid chromatography system; column: Supersil AQ-C18 (4.6 mm × 250 mm, 5 μm); detection wavelength: 200 nm; column temperature: 35 ℃; flow rate: 0.8 mL / min; injection volume: 20 μL;

[0145] Mobile phase: 0.05% phosphoric acid aqueous solution (A) – acetonitrile (B), gradient elution:

[0146]

[0147] The HPLC results of the extracts from Inula japonica in Examples 1, 2, and 3 are shown in the figures below. Figures 1-3 Among them, 1 is rutin; 2 is piperidine; and 3 is quercetin.

[0148] Experimental Example 2 Study on the anti-PTSD-like effect of Inula japonica extract in this invention

[0149] Fifty male SPF-grade C57 mice weighing 18–20 g (purchased from SPF (Beijing) Laboratory Animal Technology Co., Ltd.) were selected. After one week of acclimatization, the mice were randomly divided into 5 groups of 10 mice each, based on their weight.

[0150] Solvent group: Gavage with an equal volume of water (20 mL / kg);

[0151] Model group: administered an equal volume of water (20 mL / kg) by gavage;

[0152] Experimental group 1: Gavage administration of Inula japonica extract (500 mg / kg) as described in Example 1;

[0153] Experimental group 2: Gavage administration of Inula japonica extract (500 mg / kg) as described in Example 2;

[0154] Experimental group 3: Gavage administration of Inula japonica extract (500 mg / kg) as described in Example 3.

[0155] Before administration on day 1, experimental animals in the model group and experimental groups 1-3 were placed in an electric shock chamber for free exploration (adaptation period). After this period, they were given an inescapable 2-second foot shock (0.8 mA, lasting 2 seconds). The adaptation period and electric shock were repeated 5 times in total, with each shock occurring 120 seconds apart. The solvent group was placed in the electric shock chamber for the same duration but without any stimulation. After administration on day 7, the mice in each group were placed back in the device that had received the electric shock for environmental replication without any stimulation. The time of stiffness within 300 seconds was recorded.

[0156] See results Figure 4 Compared with the solvent group, the model group showed a significant difference (**P < 0.01); compared with the model group, the experimental group 2 showed a significant difference (##P < 0.01).

[0157] Experimental Example 3 Study on the anti-PTSD-like effect of the Inula japonica composition of this invention

[0158] Fifty male SPF-grade C57 mice weighing 18–20 g (purchased from SPF (Beijing) Laboratory Animal Technology Co., Ltd.) were selected. After one week of acclimatization, the mice were randomly divided into 5 groups of 10 mice each, based on their weight.

[0159] Solvent group: Gavage with an equal volume of water (20 mL / kg);

[0160] Model group: Gavage with an equal volume of water (20 mL / kg);

[0161] Positive control group: Sertraline (15 mg / kg, commercially available, prepared with water) was administered by gavage.

[0162] Experimental group 1: Inula japonica extract (500 mg / kg) from Example 2 was administered by gavage.

[0163] Experimental group 2: Composition of Example 4 administered by gavage (80 mg / kg).

[0164] Before administration on day 1, experimental animals in the model group, experimental groups 1-2, and the positive control group were placed in an electric shock chamber for free exploration (adaptation period). After this period, they were given an inescapable 2-second foot shock (0.8 mA, lasting 2 seconds). The adaptation period and electric shock were repeated 5 times in total, with each shock occurring 120 seconds apart. The solvent group was placed in the electric shock chamber for the same duration but without any stimulation. After administration on day 7, the mice in each group were placed back in the device that had received the electric shock for environmental replication without any stimulation. The time of arrest within 300 seconds was recorded.

[0165] See results Figure 5Compared with the solvent group, the model group showed a significant difference (**P < 0.01). Compared with the model group, the positive control group (###P < 0.001), experimental group 1 (#P < 0.05), and experimental group 2 (###P < 0.001) showed significant differences.

[0166] The above description of the embodiments is only for the purpose of helping to understand the method and core ideas of the present invention. It should be noted that those skilled in the art can make several improvements and modifications to the present invention without departing from the principles of the present invention, and these improvements and modifications also fall within the protection scope of the claims of the present invention.

Claims

1. A composition of Inula japonica, the preparation of which comprises the following steps: (1) Add water to the extract to be purified according to the weight-volume ratio of Inula japonica extract to water of 1:10-20, and disperse by ultrasonication to obtain a suspension. (2) Add petroleum ether to the prepared suspension at a volume ratio of 1-3:1, perform ultrasonic extraction, remove the petroleum ether layer, and retain the aqueous layer. (3) Repeat the ultrasonic extraction operation in step (2) 1-3 times, dilute the aqueous layer with water, disperse by ultrasonication, filter, and collect the filtrate; (4) Load the filtrate onto macroporous resin and allow it to stand for 10-18 hours to adsorb. Collect the effluent and recycle it for further loading. (5) Elute sequentially with deionized water, 10% ethanol solution, 20% ethanol solution, 30% ethanol solution and 50% ethanol solution. Collect the eluent eluted with 30% ethanol solution and the eluent eluted with 50% ethanol solution, concentrate and dry to obtain the final product.

2. The composition according to claim 1, wherein the ultrasonic conditions are ultrasonication at 40-50°C for 30-40 minutes and the ultrasonic frequency is 30-40 kHz.

3. The composition according to any one of claims 1-2, wherein in step (3) water is added to dilute it by 1-3 times.

4. The composition according to any one of claims 1-3, wherein the effluent in step (4) is circulated and loaded 5-10 times.

5. The composition according to any one of claims 1-4, wherein in step (4), the flow rate of the filtrate through the resin is 1-3 column volumes / h.

6. The composition according to any one of claims 1-5, wherein the amount of deionized water, 10% ethanol solution, 20% ethanol solution, 30% ethanol solution and 50% ethanol solution added in step (5) is 2-5 times the column volume, preferably 3-4 times the column volume.

7. A method for preparing the Inula japonica composition according to any one of claims 1-6, comprising the following steps: (1) Add water to the extract to be purified according to the weight-volume ratio of Inula japonica extract to water of 1:10-20, and disperse by ultrasonication to obtain a suspension. (2) Add petroleum ether to the prepared suspension at a volume ratio of 1-3:1, perform ultrasonic extraction, remove the petroleum ether layer, and retain the aqueous layer. (3) Repeat the ultrasonic extraction operation in step (2) 1-3 times, dilute the aqueous layer with water, disperse by ultrasonication, filter, and collect the filtrate; (4) Load the filtrate onto macroporous resin and allow it to stand for 10-18 hours to adsorb. Collect the effluent and recycle it for further loading. (5) Elute sequentially with deionized water, 10% ethanol solution, 20% ethanol solution, 30% ethanol solution and 50% ethanol solution. Collect the eluent eluted with 30% ethanol solution and the eluent eluted with 50% ethanol solution, concentrate and dry to obtain the final product.

8. An extract of Inula japonica, the preparation of which includes the following steps: (S-1) Add water to Inula japonica at a material-to-liquid ratio of 1:5–1:20 (g / mL), soak for 0.5–4 h, then heat to 30–100℃ and extract for 0.5–5 h. Filter and collect the filtrate and residue separately. (S-2) Repeat the extraction of the filter residue obtained in step (S-1) 1–3 times using the same method, and combine the filtrates obtained from each extraction; (S-3) Combine all the filtrates from steps (S-1) and (S-2), centrifuge at 2000–4000 rpm for 5–30 min, collect the supernatant, concentrate under reduced pressure to a crude drug concentration of 1:0.5–1:5 (g / mL), and dry to obtain Inula japonica extract.

9. A pharmaceutical composition comprising any one or a combination of the Inula japonica composition as claimed in any one of claims 1-6, the Inula japonica extract as claimed in claim 8, and a pharmaceutically acceptable carrier.

10. The use of any one or a combination of the Inula japonica composition of any one of claims 1-6, the Inula japonica extract of claim 8, and the Inula japonica pharmaceutical composition of claim 9 in the preparation of anti-stress articles.

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