WO2022022648A1 - 一种具有缓解精神及抗抑郁功效的中药组合物及其制备方法 - Google Patents

一种具有缓解精神及抗抑郁功效的中药组合物及其制备方法 Download PDF

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WO2022022648A1
WO2022022648A1 PCT/CN2021/109326 CN2021109326W WO2022022648A1 WO 2022022648 A1 WO2022022648 A1 WO 2022022648A1 CN 2021109326 W CN2021109326 W CN 2021109326W WO 2022022648 A1 WO2022022648 A1 WO 2022022648A1
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extract
group
water
add
xiaoyao
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PCT/CN2021/109326
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French (fr)
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闫凯境
叶正良
章顺楠
张文生
郑永锋
范立君
杜芃
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天士力医药集团股份有限公司
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Priority to EP21848960.7A priority Critical patent/EP4190342A1/en
Publication of WO2022022648A1 publication Critical patent/WO2022022648A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/23—Apiaceae or Umbelliferae (Carrot family), e.g. dill, chervil, coriander or cumin
    • A61K36/233—Bupleurum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/06—Fungi, e.g. yeasts
    • A61K36/07—Basidiomycota, e.g. Cryptococcus
    • A61K36/076—Poria
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/23—Apiaceae or Umbelliferae (Carrot family), e.g. dill, chervil, coriander or cumin
    • A61K36/232—Angelica
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/25—Araliaceae (Ginseng family), e.g. ivy, aralia, schefflera or tetrapanax
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/28—Asteraceae or Compositae (Aster or Sunflower family), e.g. chamomile, feverfew, yarrow or echinacea
    • A61K36/284—Atractylodes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/48—Fabaceae or Leguminosae (Pea or Legume family); Caesalpiniaceae; Mimosaceae; Papilionaceae
    • A61K36/484—Glycyrrhiza (licorice)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/65—Paeoniaceae (Peony family), e.g. Chinese peony
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/71—Ranunculaceae (Buttercup family), e.g. larkspur, hepatica, hydrastis, columbine or goldenseal
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/24—Antidepressants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
    • A61K2236/30—Extraction of the material
    • A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
    • A61K2236/331—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones using water, e.g. cold water, infusion, tea, steam distillation, decoction
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
    • A61K2236/30—Extraction of the material
    • A61K2236/39—Complex extraction schemes, e.g. fractionation or repeated extraction steps

Definitions

  • the application belongs to the field of traditional Chinese medicine, and specifically relates to, but is not limited to, a traditional Chinese medicine composition with the effects of relieving spirit and anti-depression, and a preparation method and application thereof.
  • Depression is a common mental illness with depression as the main symptom caused by various reasons. With the development of society, people's life and work pressures increase, and people's mental pressures are also increasing, resulting in an upward trend in the incidence of depression, and its harmfulness has attracted more and more attention from the medical and health circles. Due to the many causes of depression and the complex pathogenesis, the diagnosis and recognition rate of depression is very low.
  • the drugs for the treatment of depression on the market are mainly chemical drugs, and the antidepressant effect is not ideal, and there are problems such as high price and large side effects.
  • Chinese herbal medicine has the advantages of less toxic and side effects, less adverse reactions and long-lasting effect. Therefore, finding ideal antidepressant drugs from Chinese herbal medicine is a hot research topic in recent years.
  • Xiaoyaosan comes from the "Taiping Huimin He Ji Ju Prescription" in the Song Dynasty. Experimental studies and clinical practice have proved that it has a definite antidepressant effect.
  • Xiaoyao Powder is composed of Bupleurum, Angelica, Atractylodes, Paeonia lactiflora, Poria, Peppermint, Ginger and Licorice. of commonly used prescriptions. According to the distribution of monarchs, ministers and envoys, Bupleurum, Angelica and Baishao are the monarch medicines, Atractylodes, Poria and ginger are the ministers medicines, Mint is the assistant, and Zhigancao is the envoy.
  • the eight herbs of Xiaoyao San have different pharmacological effects. Among them, Bupleurum mainly soothes the liver and relieves stagnation, while Angelica and Baishao nourish the blood and soften the liver. These three herbs cooperate with each other to nourish and harmonize the liver. Atractylodes, Poria and Ginger can be used together. Invigorating the spleen, peppermint can help Bupleurum to disperse, and Zhigancao can nourish qi and invigorate the middle, and reconcile various medicines.
  • Chinese patents CN108653405A and CN107625813A disclose a traditional Chinese medicine composition composed of 6 traditional Chinese medicines. 20-60% of white peony root, 8-20% of mint, and 8-20% of licorice root; the preparation method includes the following steps: 1) adding 60-90% ethanol 6-10 times the weight of the component medicinal materials to extract twice , 1-3h each time, filter, combine the filtrates, and concentrate to obtain alcohol-extracted paste; 2) Add 6-10 times the weight of water to extract the drug residues twice, 1-3h each time, filter, combine the filtrates, and concentrate to obtain Water-extracting clear paste, add ethanol to the water-extracting clear paste to make the alcohol content reach 60-80%, let stand for 24 hours, take the supernatant and concentrate to clear paste; 3) Combine clear paste and alcohol-extracting paste, mix After homogenization, it is concentrated to a thick paste, dried under reduced pressure in a microwave oven, and pulverized through an 80-mesh sieve to obtain the traditional Chinese medicine extract.
  • Chinese patent CN101732427A discloses a pharmaceutical composition consisting of "Bupleurum chinensis powder, Chinese angelica extract, white peony root extract, Atractylodes macrocephala extract, Poria cocos extract, licorice extract, peppermint extract”.
  • Chinese patent CN102813906A discloses a pharmaceutical composition consisting of "Bupleurum, Angelica, Fried Atractylodes, Zhigancao, Mint, Dried Ginger".
  • Chinese patent CN104644776A discloses a traditional Chinese medicine powder for treating irregular menstruation prepared from "Bupleurum chinensis, Angelica root, Poria, Radix Paeoniae Alba, Atractylodes Rhizoma, Licorice".
  • Chinese patent CN108653405A discloses that the alcohol-extracted paste is obtained by first merging alcohol extraction, then the water-extracted paste is added, and alcohol is added to the water-extracted paste to obtain the paste, and the alcohol-extracted paste and the purified paste obtained for the second time Preparation method of paste combination.
  • the extraction of volatile oils by steam distillation and other methods is also widely used in the preparation of Xiaoyao products.
  • Chinese patent CN101732427A discloses that the other components in the formula except Bupleurum chinensis are combined with water and the volatiles are retained, ethanol is added to the water-extracted thick paste and filtered twice, and the filtrate is combined with the volatiles and Bupleurum fine powder. Preparation.
  • Chinese Patent Publication CN104189833A discloses: 1) steam distillation of four medicinal materials of Bupleurum, Angelica Root, Chaobaizhu and Mint to obtain volatile oil, 2) Use cyclodextrin to treat the volatile oil to obtain inclusion compound, 3) Baishao, Mizhi The licorice root, Poria cocos and the medicinal residues obtained by extraction in step 1) are combined with water extraction, and volatile oil inclusion compounds are added to obtain the preparation method of the tablet.
  • Chinese patent publication CN102908600A discloses: 1) extracting volatile oil from Bupleurum, ginger, mint, and angelica, 2) extracting the extracted medicinal residues with fried Atractylodes and Poria, and concentrating to obtain a thick paste, 3) Paeonia lactiflora, four points One equivalent of Zhigancao and the remaining equivalent of Angelica sinensis are crushed into medicinal powder, 4) the remaining amount of Zhigancao is made into extract, and 5) the preparation method of combining the obtained pills.
  • the present application provides a traditional Chinese medicine composition with the functions of relieving spirit and antidepressant, and a preparation method and application thereof.
  • the inventors of the present application have conducted meticulous research on the formulations of the prior art, and have used modern science and technology to study the antidepressant mechanism of Xiaoyaosan and its similar formulations, and screened out Xiaoyaosan and its similar formulations to have a better antidepressant effect.
  • the present application has the following characteristics: (1) After the inventors of the present application have conducted research on various formulas, the selected formulas are simpler and have lower costs. Wanquan recipe (Qiwei, "Chinese Pharmacopoeia” 2015 edition No. 1354-1355) and the recipe with mint added (Liuwei) (CN108653405A, CN107625813A) more superior antidepressant effect; (2) the preparation process only uses water as a solvent to carry out Extraction, the preparation process is simpler, safer and more controllable, and the cost is lower; (3) the inventor of the present application adopts the extraction process of grouping, which avoids the interference of other components on the quantification of ferulic acid in Angelica sinensis.
  • Each medicine can be quality controlled without mutual interference.
  • Bupleurum and Angelica are separated to avoid the interference of Bupleurum components on the quantification of ferulic acid in Angelica; each group of extracts contains at least one stable index component to characterize the process stability from the extract to the preparation process; The bran fried Atractylodes and Angelica sinensis with volatile components are grouped together.
  • the aromatic water is collected during the extraction process, and added to the extract at the end of the concentrated ointment, so that the overall extract is close to the traditional application of water decoction.
  • the present application provides a traditional Chinese medicine composition with relieving spirit and antidepressant effects
  • the traditional Chinese medicinal composition is composed of Bupleurum radix, Paeonia lactiflora Radix, bran fried Atractylodes Rhizoma, Angelica sinensis, Zhigancao and optionally with or without the addition of
  • the traditional Chinese medicine crude drug formula in the weight portion of Poria cocos is prepared, and the formula that does not add Poria cocos is Formula 1:
  • the present application provides a preparation method of the above-mentioned traditional Chinese medicine composition.
  • the preparation method includes the following steps: weighing the medicinal materials according to the composition ratio, adding 4-7 times the weight of water, extracting 1-3 times, each time time for 1-3 hours, filter after extraction, combine the filtrates from the two extractions, let stand for more than 4 hours, take the supernatant, and concentrate to obtain the extract; take the extract as the active ingredient of the medicine, and add medicinal excipients if necessary to prepare a medicine Preparation of traditional Chinese medicine composition.
  • the application also provides a pharmaceutical preparation Chinese medicine composition (referred to as Chinese medicine preparation composition), which is an oral preparation or an injection.
  • the traditional Chinese medicine composition can be in any ingestible pharmaceutical form: such as: tablets, sugar-coated tablets, film-coated tablets, enteric-coated tablets, capsules, hard capsules, soft capsules preparations, oral liquids, buccal preparations, granules, granules, pills, powders, ointments, pills, suspensions, powders, solutions, injections, suppositories, ointments, plasters, creams, sprays, drops or patch.
  • the prescription and preparation process, the antidepressant efficacy of Xiaoyaosan varies with the formula and preparation process, and the obtained active ingredients that exert antidepressant efficacy
  • the composition is very different.
  • the present application adopts a specific extraction process according to specific medicinal materials and proportions to obtain different compositions with antidepressant effects.
  • Figure 1 The specificity of the analysis method for ferulic acid content in the combined extraction of 5 medicinal materials is unqualified, wherein E03 is Example 14 of the application.
  • Figure 2 The specificity of the analytical method for the content of ferulic acid in Xiaoyaodang Baigan extract is qualified.
  • Figure 3 Graph of changes in glycyrrhizic acid content.
  • the present application provides a traditional Chinese medicine composition with relieving spirit and antidepressant effects
  • the traditional Chinese medicinal composition is composed of Bupleurum radix, Paeonia lactiflora Radix, bran fried Atractylodes Rhizoma, Angelica sinensis, Zhigancao and optionally with or without the addition of
  • the traditional Chinese medicine crude drug formula in the weight portion of Poria cocos is prepared, and the formula that does not add Poria cocos is Formula 1:
  • the formula 1 is: Bupleurum 15-25, Baishao 15-25, Bran fried Atractylodes 15-25, Angelica 15-25, Zhigancao 15-20;
  • the formula 2 is: Bupleurum 15-25, Baishao 15-25, Bran fried Atractylodes 15-25, Angelica 15-25, Poria 15-25, Zhigancao 15-20.
  • the formula 1 is: Bupleurum 20, Baishao 20, Bran fried Atractylodes 20, Angelica 20, Zhigancao 16; wherein the formula 2 is: Bupleurum 20, White peony root 20, bran fried Atractylodes 20, angelica 20, tuckahoe 20, licorice 16.
  • the traditional Chinese medicine composition of the formula 1 includes 9 parts of Xiaoyaochaibai extract by dry weight, and the Xiaoyaochaibai extract is extracted from 10-30 parts of Bupleurum and 10-30 parts of Paeonia lactiflora Obtained; 32 parts of xiaoyaodangbaigan extract by dry weight, the xiaoyaodangbaigan extract is extracted from bran fried Atractylodes 15-30, Angelica 10-30, Zhigancao 15-24; wherein the Xiaoyao The dry content of saikosaponin B2 in the Chaibai extract is 0.63-10.0 mg/g, and the dry content of paeoniflorin is 9.4-115.9 mg/g; the dry content of glycyrrhizic acid in the Xiaoyaodangbaigan extract is 1.4-26.1 mg/g. g. The dry content of ferulic acid is 0.13-0.91 mg
  • the dry content of saikosaponin B2 in the Xiaoyao Chaibai extract is 0.94-6.8 mg/g, and the dry content of paeoniflorin is 17.6-110.9 mg/g;
  • the dry content of glycyrrhizic acid is 6.9-26.1 mg/g, the dry content of ferulic acid is 0.16-0.79 mg/g, and the dry content of atractylide III is 0.0047-0.25 mg/g.
  • the dry weight in this application refers to the weight after drying the sample, that is, the weight after drying.
  • the present application provides a preparation method of the above-mentioned traditional Chinese medicine composition.
  • the preparation method includes the following steps: weighing the medicinal materials according to the composition ratio, adding 4-7 times the weight of water, extracting 1-3 times, each time time for 1-3 hours, filter after extraction, combine the filtrates from the two extractions, let stand for more than 4 hours, take the supernatant, and concentrate to obtain the extract; take the extract as the active ingredient of the medicine, and add medicinal excipients if necessary to prepare a medicine Preparation of traditional Chinese medicine composition.
  • the preparation method includes the steps of: weighing the medicinal materials according to the formula, adding 6-7 times the weight of water, extracting for 2-3 hours, filtering, and adding 5-6 times the weight of the medicinal residues to the medicinal residues.
  • the auxiliary material is prepared into a traditional Chinese medicine composition of a pharmaceutical preparation.
  • the preparation method of the traditional Chinese medicine composition of described formula 1 comprises the steps:
  • the preparation method of the traditional Chinese medicine composition of described formula 1 comprises the steps:
  • the inventor of the present application further found that the quantification of ferulic acid in Angelica was interfered by some components of Bupleurum, and the specificity was unqualified when the remaining five medicinal materials were combined with water extraction after the mint and Poria recipes were reduced, as shown in Figure 1 shown.
  • the inventors designed a good extraction process, and by grouping extraction, the interference of other components on the quantification of ferulic acid in Angelica was avoided, as shown in Figure 2.
  • Group extraction, using the extract as medicine not only the efficacy is not reduced, but its quality is effectively controlled.
  • the saikosaponin components that can be detected in the saikosaponin are saikosaponin A and saikosaponin D.
  • saikosaponin D is converted into B2.
  • saikosaponin B2 can be detected in the extract and dried. highest content.
  • the present application also provides a pharmaceutical preparation Chinese medicine composition (referred to as a Chinese medicine preparation composition), which can be an oral preparation or an injection.
  • a pharmaceutical preparation Chinese medicine composition referred to as a Chinese medicine preparation composition
  • the oral preparation is selected from one of capsules, tablets, dropping pills, granules, concentrated pills, oral liquids and mixtures.
  • the injection is selected from one of injection, freeze-dried powder injection and water injection.
  • the traditional Chinese medicine preparation composition of the present application is in any available pharmaceutical form: such as: tablets, sugar-coated tablets, film-coated tablets, enteric-coated tablets, capsules, hard capsules, soft Capsules, oral liquids, buccal preparations, granules, granules, pills, powders, ointments, pills, suspensions, powders, solutions, injections, suppositories, ointments, plasters, creams, sprays, drops dose or patch.
  • any available pharmaceutical form such as: tablets, sugar-coated tablets, film-coated tablets, enteric-coated tablets, capsules, hard capsules, soft Capsules, oral liquids, buccal preparations, granules, granules, pills, powders, ointments, pills, suspensions, powders, solutions, injections, suppositories, ointments, plasters, creams, sprays, drops dose or patch.
  • the traditional Chinese medicine preparation composition of the present application is in the form of a unit dose pharmaceutical preparation.
  • the unit dose of the medicament may contain 0.1-1000 mg of the traditional Chinese medicinal composition of the present application, and the rest are pharmaceutically acceptable auxiliary materials.
  • the pharmaceutically acceptable adjuvants can be 0.01-99.99% by weight of the total weight of the formulation.
  • the dosage of the traditional Chinese medicine preparation of the present application is determined according to the patient's condition, such as 1-3 times a day, 1-20 tablets at a time, etc.
  • the preparation for oral administration may contain commonly used pharmaceutical excipients, such as but not limited to binders, fillers, diluents, compressed tablets, lubricants, disintegrants, colorants, Flavoring and wetting agents, if necessary, to coat tablets.
  • pharmaceutical excipients such as but not limited to binders, fillers, diluents, compressed tablets, lubricants, disintegrants, colorants, Flavoring and wetting agents, if necessary, to coat tablets.
  • Suitable fillers include cellulose, mannitol, lactose and other similar fillers.
  • Suitable disintegrants include starch, polyvinylpyrrolidone and starch derivatives such as sodium starch glycolate.
  • Suitable lubricants include, for example, magnesium stearate.
  • Suitable pharmaceutically acceptable humectants include sodium lauryl sulfate.
  • the traditional Chinese medicine preparation composition of the present application can be prepared as a solid oral composition by common methods such as mixing, filling, and tableting. Repeated mixing allows the active to be distributed throughout those compositions where large amounts of fillers are used.
  • Oral liquid preparations can be in the form of, for example, aqueous or oily suspensions, solutions, emulsions, syrups or elixirs, or can be a dry product for constitution with water or other suitable vehicle before use.
  • Such liquid preparations may contain conventional additives such as suspending agents, for example sorbitol, syrup, methylcellulose, gelatin, hydroxyethylcellulose, carboxymethylcellulose, aluminum stearate gel or hydrogenated edible fats, Emulsifiers such as lecithin, sorbitan monooleate or acacia; non-aqueous carriers (which may include edible oils) such as almond oil, fractionated coconut oil, oily esters such as glycerol esters, propylene glycol or ethanol; preservatives agents such as methylparaben or propylparaben or sorbic acid and, if desired, conventional flavoring or coloring agents.
  • suspending agents for example sorbitol, syrup, methylcellulose, gelatin, hydroxy
  • liquid unit dosage forms are prepared containing the active substance of the application and a sterile carrier. Depending on the carrier and concentration, the active can be suspended or dissolved. Solutions are usually prepared by dissolving the active substance in a carrier, filter sterilizing before filling into a suitable vial or ampoule, and sealing. Adjuvants such as a local anesthetic, preservatives and buffering agents can also be dissolved in the carrier. To improve its stability, the composition can be frozen after filling into a vial and the water removed under vacuum.
  • Bupleurum 10kg, white peony root 10kg, bran fried Atractylodes 15kg, angelica 10kg, licorice 15kg. Prepared as in Example 16. Weigh the medicinal materials according to the formula ratio, add 6 times the weight of water, extract for 2 hours, filter, add 5 times the weight of water to the medicinal residues, extract for 1 hour, filter, combine the filtrates from the two extractions, and stand at room temperature for more than 4 hours. The supernatant is taken, and the concentration end point is controlled at 68% to 72% sugar content to obtain a traditional Chinese medicine extract.
  • the supernatant is taken and concentrated to a sugar content of 65% to 70% to obtain an extract.
  • the supernatant is taken and concentrated to a sugar content of 65% to 70% to obtain an extract.
  • the supernatant is taken and concentrated to a sugar content of 65% to 70% to obtain an extract.
  • the supernatant is taken and concentrated to a sugar content of 65% to 70% to obtain an extract.
  • the supernatant is taken and concentrated to a sugar content of 65% to 70% to obtain an extract.
  • the solution is concentrated to 65% to 70% sugar content to obtain the extract.
  • the folded dry content in the following content determination extract content/extract dry residue, the extract dry residue is equal to the solid content, and the detection method refers to the European Pharmacopoeia ⁇ 2.8.16>.
  • Chromatographic conditions Waters UPLC chromatograph, chromatographic column: BEH C18 (2.1mm*100mm, 1.7 ⁇ m) chromatographic column, the mobile phase is acetonitrile (A)-water (B), the gradient elution is carried out according to the following table, the detection wavelength is 254nm, The column temperature was 30°C.
  • 60% methanol-0.5mol/L NaOH solution take 2g of sodium hydroxide, add water to dissolve it into 100ml, that is, 0.5mol/L NaOH solution, take 60ml of methanol, add 0.5mol/L NaOH solution to make 100ml, mix well, that is, have to. (According to actual needs, the amount of each reagent and reagent added can be adjusted proportionally)
  • the dry content of saikosaponin B2 is 0.63 ⁇ 10.0mg/g
  • test product the peak area of the test product
  • reference substance reference substance solution concentration, unit mg/ml
  • a reference substance peak area of reference substance
  • m weighed sample size of the test sample, in g
  • 0.05% phosphoric acid aqueous solution take 0.5ml of phosphoric acid, add ultrapure water to make 1000ml, that is. (According to actual needs, the amount of each reagent added can be adjusted proportionally)
  • 30% methanol solution take 30ml of methanol, add water to make 100ml, and mix evenly. (According to actual needs, the amount of each reagent added can be adjusted proportionally)
  • the dry content of paeoniflorin is 9.4 ⁇ 115.9mg/g
  • test product the peak area of the test product
  • reference substance reference substance solution concentration, unit mg/ml
  • a reference substance peak area of reference substance
  • m weighed sample size of the test sample, in g
  • paeoniflorin dry the dry content of paeoniflorin, in mg/g;
  • 0.05% phosphoric acid aqueous solution take 0.5 ml of phosphoric acid, add ultrapure water to make 1000 ml, and get it. (According to actual needs, the amount of each reagent added can be adjusted proportionally)
  • 30% methanol solution take 30ml of methanol, add water to make 100ml, and mix evenly. (According to actual needs, the amount of each reagent added can be adjusted proportionally)
  • the dry content of glycyrrhizic acid is 1.4 ⁇ 26.1mg/g
  • test product the peak area of the test product
  • reference substance reference substance solution concentration, unit mg/ml
  • a reference substance peak area of reference substance
  • m weighed sample size of the test sample, in g
  • glycyrrhizic acid dry content of glycyrrhizic acid, unit mg/g;
  • 0.05% phosphoric acid aqueous solution take 0.5 ml of phosphoric acid, add ultrapure water to make 1000 ml, and get it. (According to actual needs, the amount of each reagent added can be adjusted proportionally)
  • 30% methanol solution take 30ml of methanol, add water to make 100ml, and mix evenly. (According to actual needs, the amount of each reagent added can be adjusted proportionally)
  • the dry content of ferulic acid is 0.13 ⁇ 0.91mg/g
  • test product the peak area of the test product
  • reference substance reference substance solution concentration, unit mg/ml
  • a reference substance peak area of reference substance
  • m weighed sample size of the test sample, in g
  • ferulic acid dry the dry content of ferulic acid, in mg/g
  • 0.05% phosphoric acid aqueous solution take 0.5 ml of phosphoric acid, add ultrapure water to make 1000 ml, and get it. (According to actual needs, the amount of each reagent added can be adjusted proportionally)
  • 30% methanol solution take 30ml of methanol, add water to make 100ml, and mix evenly. (According to actual needs, the amount of each reagent added can be adjusted proportionally)
  • the dry content of atractylide III is 0.0038 ⁇ 0.30mg/g
  • test product the peak area of the test product
  • reference substance reference substance solution concentration, unit mg/ml
  • a reference substance peak area of reference substance
  • m weighed sample size of the test sample, in g
  • Atractylidene Lactone III the dry content of Atractylodes Lactone III, in mg/g;
  • CUMS Chronic unpredictable mild stress
  • CUMS Chronic unpredictable mild stress
  • rats were induced to produce many abnormal behavioral symptoms similar to those in patients with depression.
  • weight change, sugar water consumption percentage, and open-box test exercise score are the main behavioral evaluation indicators of the depression model, which can reflect the symptoms of clinical depression, such as loss of appetite, anhedonia, autonomous activity, and decreased curiosity about the fresh environment. .
  • the pathogenesis of depression is complex, and currently widely recognized and in-depth hypotheses include the monoamine neurotransmitter hypothesis, the hypothalamic-pituitary-adrenal (HPA) axis hyperfunction hypothesis, and the neuroplasticity hypothesis.
  • the monoamine neurotransmitter hypothesis as the most classic hypothesis of depression, believes that the occurrence of depression is mainly due to monoamine neurotransmitters such as serotonin (5-HT), dopamine (DA) in the central nervous system of the brain ) and norepinephrine (NE) deficiency, which is also the target of most antidepressants in clinical practice; hyperfunction of the hypothalamic-pituitary-adrenal (HPA) axis can lead to abnormal levels of corticosterone (CORT) in the body
  • the neuroplasticity hypothesis is that brain-derived neurotrophic factor (BDNF) is involved in regulating the survival, growth, differentiation and apoptosis of nerve cells.
  • BDNF brain-derived neurotrophic factor
  • Group A modified Xiaoyao Recipe
  • Group C Xiaoyao Recipe
  • Group D Example 16 of this application
  • Group E Example 28 of this application
  • Group F extracts prepared with reference to Chinese patent CN108653405A
  • Table 1 The formulations were prepared by Tasly Pharmaceutical Group Co., Ltd.; Fluoxetine Hydrochloride (Flu) was produced by Eli Lilly Suzhou Pharmaceutical Co., Ltd.; Plasma corticosterone (CORT) ELISA kit, neurotrophic factor (BDNF) ELISA kit, 5- Serotonin (5-HT) ELISA kit, dopamine (DA) ELISA kit, and norepinephrine (NE) ELISA kit were purchased from R&D Systems in the United States; physiological saline was purchased from Shijiazhuang Fourth Pharmaceutical Co., Ltd.
  • BDNF neurotrophic factor
  • 5- Serotonin 5-HT
  • DA dopamine
  • NE norepinephrine
  • Open box (100cm ⁇ 100cm ⁇ 50cm, self-made), LTD0114 bullet head light (Zhejiang Anhua Safety Equipment Co., Ltd.), G6805-1 therapeutic apparatus (Qingdao Xinsheng Industrial Co., Ltd., China), wooden clip, TD21001 electronic balance (Tianjin Tianma Instrument Factory), IKA T18 Ultrasonic Tissue Disruptor (Ningbo Xinzhi Biotechnology Co., Ltd.), 3K15 Refrigerated High-speed Centrifuge (Sigma Company, USA), Infinite M200Pro Multi-Function Microplate Reader (Swiss TECAN Company),- 80°C low temperature refrigerator (Thermo Technology Company, USA).
  • fluoxetine hydrochloride capsules (20 mg/capsule), remove the capsule shell, add 0.5% CMC-Na solution and grind to prepare a 1 mg/mL fluoxetine suspension.
  • the daily clinical doses of the extracts in Group A, Group C, Group D, Group E and Group F were 5.21g, 3.21g, 3.26g, 3.29g, and 2.55g, respectively, which were equivalent to 15 times the clinically equivalent dose in rats. It is 7.0305g/kg, 4.3380g/kg, 4.4040g/kg, 4.4415g/kg, 3.4425g/kg, see Table 1 for details.
  • Ointment yield (%) extract weight / medicinal material weight ⁇ 100%
  • rats After 130 SD rats were adaptively reared for 7 days, the rats were subjected to an open-box experiment in a quiet environment. The rats were screened according to the principle of similar horizontal and vertical motor scores, and those with too high or too low scores were excluded. The selected rats were randomly divided into normal group, model group, Flu group, A group, C group, D group, E group and F group, with 13 rats in each group.
  • the rats in the normal group were routinely housed with 4 rats per cage, with free access to water and food, and did not receive any other additional stimuli; the rats in the other groups were housed in a single cage and randomly received the following 2 different stress stimuli every day, and the same stimuli could not appear consecutively. , so that the rat can not predict what kind of stimulation, to avoid its adaptation.
  • CUMS stimulation factors include: restraint for 2h, ice water high platform for 2h, noise stimulation (110dB, 30min), foot shock (current 4mA, 50 times, each time interval 5s), light stimulation (100 000lx, 30min), day and night reversal for 24h, Wet bedding for 12h, and tail clipping (about 1cm from the tip of the rat tail, 2min). Models were continuously established, and the body weight gain, the percentage of sugar water consumption, and the scores of horizontal and vertical movements in the open-box experiment were measured weekly.
  • the rats in each group were subjected to sugar water consumption experiments. Three days before the experiment, sugar water adaptation was carried out. Two bottles of 1% sucrose solution were placed on the cage for 24 hours, then a bottle of 1% sucrose solution and a bottle of pure water were placed for 24 hours. The position of the water bottle was changed in the morning and afternoon. . In the formal experiment, the rats in each group were placed a bottle of 1% sucrose solution and a bottle of pure water, and the positions of the water bottles were changed in the morning and afternoon. percentage(%). The formula is as follows:
  • the rats in each group were subjected to the open-box experiment.
  • the size of the open box used in the experiment is 100cm ⁇ 100cm ⁇ 50cm (length ⁇ width ⁇ height), the inner side is black, and the bottom is divided into 100 (10cm ⁇ 10cm) small squares of the same size with white lines. Place each rat in turn in a fixed square in the center to exclude human interference, and record the activity route of the rat within 3 min. The number of small squares on the bottom surface of the experimental rat was used as the score of horizontal movement.
  • BDNF nerve growth trophic factor
  • 5-HT serotonin
  • DA dopamine
  • NE norepinephrine
  • the content of CORT in plasma and the content of BDNF, 5-HT, DA and NE in cortex and hippocampus of rats in each group were determined by enzyme-linked immunosorbent assay (ELISA).
  • the rats in each experimental group increased their body weight in different degrees with the modeling process.
  • the results are shown in Table 2.
  • the body weight increase of the rats in the model group was significantly lower than that in the normal group (P ⁇ 0.01), indicating that the combination of CUMS and the solitary feeding method has a great influence.
  • the rat's appetite led to slow weight gain, mimicking the symptoms of anorexia and weight loss in depressed patients.
  • the weight gain of the rats in each administration group was significantly higher than that in the model group (P ⁇ 0.01), indicating that each administration group could resist the weight loss of the CUMS combined with the isolated depression model rats, thereby improving the CUMS combined with the isolated depression model rats.
  • Loss of appetite, weight loss symptoms It can be seen from the table that the relationship between the weight gain of the rats in each administration group is in the order of Flu group>D group>F group>E group>C group>A group, indicating that the extract of group D improves the CUMS combined with the depression model of solitary raising.
  • the horizontal movement scores of the rats in each experimental group gradually decreased with the modeling process.
  • the results are shown in Table 4.
  • the horizontal movement scores of the rats in the model group were significantly lower than those in the normal group ( P ⁇ 0.01), indicating that CUMS combined with the solitary model method can significantly reduce the emotion and autonomic activity of rats, and simulate the symptoms of low autonomic activity in patients with depression.
  • the open-box level exercise scores of each administration group were significantly higher (P ⁇ 0.01), indicating that each administration group could effectively up-regulate the emotion and autonomous activity of CUMS combined with solitary depression model rats, thereby improving CUMS combined with depression.
  • Symptoms of low autonomic activity in orphaned depression model rats It can be seen from the table that the relationship between the horizontal exercise scores of the rats in each administration group is in the order of Flu group > D group > E group > F group > C group > A group, indicating that the extract of group D improves the CUMS combined with the depression model of solitary raising.
  • the vertical and horizontal movement scores of the rats in each experimental group gradually decreased with the modeling process.
  • the results are shown in Table 5.
  • the open box vertical movement scores of the rats in the model group were significantly lower than those in the normal group (P ⁇ 0.01), indicating that CUMS combined with the solitary model method can significantly reduce the spatial exploration ability of rats, simulating the symptoms of depression patients' reduced curiosity about the fresh environment.
  • the CORT content in the plasma of the rats in each group is shown in Table 6. Compared with the normal group, the CORT content in the plasma of the model group was significantly increased (P ⁇ 0.01), indicating that CUMS combined with the solitary model method significantly increased the CORT content in the plasma of the rats, resulting in hyperfunction of the HPA axis, thereby causing depression in the rats.
  • the contents of DA, NE, 5-HT and BDNF in the cortex of rats in each group are shown in Table 7.
  • the contents of DA, NE, 5-HT and BDNF in the cortex of the rats in the model group were significantly lower than those in the normal group (P ⁇ 0.01), indicating that CUMS combined with the solitary model method significantly reduced the levels of monoamine neurotransmitters DA and NE in the rat cortex.
  • 5-HT content and brain-derived neurotrophic factor BDNF content thereby causing depression in rats.
  • each administration group Compared with the model group, the contents of DA, NE, 5-HT and BDNF in the cortex of the rats in each administration group were significantly increased (P ⁇ 0.01), indicating that each administration group could increase the levels of monoamines in the cortex of CUMS combined with isolated depression model rats. It can increase the level of neurotransmitter, and by up-regulating the content of BDNF in the cortex, it can promote the repair and regeneration of neurons, thereby exerting an antidepressant effect.
  • the relationship between the contents of DA, NE and 5-HT in the cortex of the rats in each administration group is in the order of Flu group>D group>F group>E group>C group>A group, the rats in each administration group
  • the relationship between the content of BDNF in cortex was D group>Flu group>F group>E group>C group>A group, indicating that the antidepressant effect of the extract in group D was the best, and the antidepressant effect of the extract in group A was the worst.
  • the content of DA, NE, and 5-HT in the cortex was significantly different between group D and group E, P ⁇ 0.05. There was a significant difference in BDNF content between group D and group E, P ⁇ 0.01.
  • Table 8 shows the comparison of DA, NE, 5-HT and BDNF contents in hippocampus of rats in each group.
  • the contents of DA, NE, 5-HT and BDNF in the hippocampus of rats in the model group were significantly lower than those in the normal group (P ⁇ 0.01), indicating that CUMS combined with the solitary model method significantly reduced the levels of monoamine neurotransmitters DA and NE in the hippocampus of rats.
  • 5-HT content and brain-derived neurotrophic factor BDNF content thereby causing depression in rats.
  • the relationship between the contents of DA, NE, and 5-HT in the hippocampus of the rats in each administration group is in the order of Flu group > D group > F group > E group > C group > A group, and the rats in each administration group are in the following order.
  • the relationship between the contents of BDNF in hippocampus was D group>Flu group>F group>E group>C group>A group, indicating that the antidepressant effect of the extract of group D was the best, and the antidepressant effect of group A was the worst.
  • the content of DA and NE in the hippocampus was significantly different between group D and group E, P ⁇ 0.05; the content of 5-HT and BDNF was significantly different between group D and group E, P ⁇ 0.01.
  • CUMS Chronic mild and unpredictable stress
  • the results of the drug efficacy indexes of each group of rats are shown in Table 9. It can be seen from the table: in general, the relationship between the results of the drug efficacy indicators of the rats in each administration group is in the order of Flu group, D group>F group, E group>C group>A group, indicating that the antidepressant effect of the extract in group D is closely related to the drug in group Flu. Quite, group A extracts had the worst antidepressant effect.
  • the extracts of group A, group C, group D, group E, and F can exert antidepressant effects by resisting the hyperfunction of HPA axis, increasing the content of monoamine neurotransmitters and improving neuronal plasticity.
  • the antidepressant effect of the D group extract was comparable to that of the Flu group, and the antidepressant effect of the A group extract was the worst. There was a significant difference between group D and group E.
  • Test Example 2 Comparative data of the grouped extraction composition of the present application and the combined extraction composition
  • Group B (Example 14), Group C, Group D and Group F are the same as in Table 1 of Test Example 1.
  • fluoxetine hydrochloride capsules (20 mg/capsule), remove the capsule shell, add 0.5% CMC-Na solution and grind to prepare a 1 mg/mL fluoxetine suspension.
  • the daily clinical doses of the extracts in Group B, Group C, Group D and Group F were 3.26g, 3.37g, 3.23g and 2.72g, respectively, and the doses converted into rats were 4.4010g/ kg, 4.5495g/kg, 4.3605g/kg, 3.6720g/kg.
  • rats After 90 SD rats were adaptively reared for 7 days, the rats were subjected to an open-box experiment in a quiet environment. The rats were screened according to the principle of similar horizontal and vertical motor scores, and those with too high or too low scores were excluded. The selected rats were randomly divided into normal group, model group, Flu group, B group, C group, D group and F group, with 12 rats in each group.
  • CUMS stimulation factors include: restraint for 2h, ice water high platform for 2h, noise stimulation (110dB, 30min), foot shock (current 4mA, 50 times, each time interval 5s), 45°C oven for 10min, day and night upside down for 24h, moist pad Feed for 12h, and clip the tail (about 1cm from the tip of the rat tail, 2min). Models were continuously established, and the body weight gain, the percentage of sugar water consumption, and the scores of horizontal and vertical movements in the open-box experiment were measured weekly.
  • the rats in each group were subjected to sugar water consumption experiments. Three days before the experiment, sugar water adaptation was carried out. Two bottles of 1% sucrose solution were placed on the cage for 24 hours, then a bottle of 1% sucrose solution and a bottle of pure water were placed for 24 hours. The position of the water bottle was changed in the morning and afternoon. . In the formal experiment, the rats in each group were placed a bottle of 1% sucrose solution and a bottle of pure water, and the positions of the water bottles were changed in the morning and afternoon. percentage(%). The formula is as follows:
  • the rats in each group were subjected to the open-box experiment.
  • the size of the open box used in the experiment is 100cm ⁇ 100cm ⁇ 50cm (length ⁇ width ⁇ height), the inner side is black, and the bottom is divided into 100 (10cm ⁇ 10cm) small squares of the same size with white lines. Place each rat in turn in a fixed square in the center to exclude human interference, and record the activity route of the rat within 3 min. The number of small squares on the bottom surface of the experimental rat was used as the score of horizontal movement.
  • BDNF nerve growth trophic factor
  • 5-HT serotonin
  • DA dopamine
  • NE norepinephrine
  • the content of CORT in plasma and the content of BDNF, 5-HT, DA and NE in cortex and hippocampus of rats in each group were determined by enzyme-linked immunosorbent assay (ELISA).
  • the rats in each experimental group increased their body weight in different degrees with the modeling process.
  • the results are shown in Table 10.
  • the body weight increase of the rats in the model group was significantly lower than that in the normal group (P ⁇ 0.01), indicating that CUMS combined with the solitary model method affects the appetite of rats and leads to slow weight gain, which simulates the symptoms of loss of appetite and weight loss in patients with depression.
  • the weight gain of the rats in each administration group was significantly higher than that of the model group (P ⁇ 0.01).
  • the weight gain of the rats in the Flu group was higher than that of the D group, but there was no significant difference between the Flu group and the D group (P ⁇ 0.01).
  • P>0.05 there were significant differences in the weight gain of rats in other groups (P ⁇ 0.05), indicating that each administration group could resist the weight loss of CUMS combined with orphaned depression model rats, thereby improving CUMS combined with solitary depression. Loss of appetite and weight loss in model rats.
  • the sugar water consumption percentage of rats in each experimental group gradually decreased with the modeling process.
  • the results are shown in the sugar water consumption ratio of the rats in each group.
  • CUMS combined with the solitary model method reduces the percentage of sugar water consumption in rats, and simulates anhedonia symptoms in patients with depression.
  • the horizontal movement scores of the rats in each experimental group gradually decreased with the modeling process; the results of the horizontal movement scores of the rats in each group are shown in Table 12, and the scores of the rats in the model group were significantly lower than those in the normal group (P ⁇ 0.01), indicating that CUMS combined with the solitary model method can significantly reduce the emotion and autonomic activity of rats, simulating the symptoms of low autonomic activity in patients with depression.
  • the scores of vertical movement in the open box of each administration group were significantly higher (P ⁇ 0.05), and the scores of vertical movement in the open box of the rats in other groups were significantly different (P ⁇ 0.05), indicating that the scores of the rats in each administration group were significantly higher than those of the model group (P ⁇ 0.05). It can improve the symptoms of reduced curiosity about fresh environment in CUMS combined with isolated depression model rats. It can be seen from Table 14 that the relationship between the vertical movement scores of the rats in each administration group is in the order of Flu group > D group > B group > F group > C group, indicating that the extract of group D improves the effect of CUMS combined with isolated depression model rats on the fresh environment.
  • Corticosterone is a glucocorticoid, and high concentrations of corticosterone bind to its receptors to produce neurotoxicity, causing neuronal damage in the hippocampus, leading to hyperfunction of the hypothalamic-pituitary-adrenal (HPA) axis, thereby causing depression.
  • HPA hypothalamic-pituitary-adrenal
  • the content of CORT in plasma of rats in each group is shown in Table 15. Compared with the normal group, the CORT content in the plasma of the model group was significantly increased (P ⁇ 0.01), indicating that CUMS combined with the solitary model method significantly increased the CORT content in the plasma of the rats, resulting in hyperfunction of the HPA axis, thereby causing depression in the rats.
  • the CORT content in the plasma of the rats in each administration group was significantly decreased (P ⁇ 0.01), and the CORT content in the plasma of the rats in other groups was significantly different (P ⁇ 0.05), indicating that each administration group could Improve the HPA axis hyperfunction of CUMS combined with the solitary model of depressed rats, thereby exerting an antidepressant effect.
  • Table 15 the relationship between the CORT content in the plasma of the rats in each administration group is in the order of Flu group ⁇ D group ⁇ B group ⁇ F group ⁇ C group, indicating that the antidepressant effect of the extract in group D is second only to the drug in group Flu, and group C The group extract had the least antidepressant effect. There was a significant difference between group D and group B, P ⁇ 0.05.
  • Dopamine (DA), norepinephrine (NE), and serotonin (5-HT) are monoamine neurotransmitters in the central nervous system of the brain, and brain-derived neurotrophic factor (BDNF) is a neurotrophic Decreased levels of a supportive protein can lead to depression.
  • BDNF brain-derived neurotrophic factor
  • the contents of DA, NE, 5-HT and BDNF in the cortex of rats in each group are shown in Table 16.
  • Table 17 shows the comparison of the contents of DA, NE, 5-HT and BDNF in the hippocampus of rats in each group.
  • the contents of DA, NE, 5-HT and BDNF in the cortex and hippocampus of the rats in the model group were significantly lower than those in the normal group (P ⁇ 0.01), indicating that CUMS combined with the solitary model method significantly reduced monoamine neurotransmitters in the cortex and hippocampus of rats.
  • the content of DA, NE, 5-HT and the content of brain-derived neurotrophic factor BDNF can cause depression in rats.
  • the relationship between the content of DA in hippocampus of rats in each administration group was in the order of Flu group>D group>B group>F group>C group, and there was a significant difference between D and B, P ⁇ 0.01.
  • the above shows that the antidepressant effect of the D group extract is second only to the Flu group, and the C group extract has the worst antidepressant effect.
  • the content of 5-HT in the cortex and hippocampus of the rats in each administration group was significantly higher than that in the model group (P ⁇ 0.05). There was no significant difference in the content of 5-HT in the hippocampus of the rats and the F group (P>0.05), and there were significant differences in the content of 5-HT in the cortex and hippocampus of the rats in other groups (P ⁇ 0.05), indicating that each administration group Both of them can increase the 5-HT content in the cortex and hippocampus of CUMS combined with isolated depression model rats, thereby exerting an antidepressant effect.
  • the behavioral indicators (body weight change, sugar water consumption percentage, open box test exercise score) of rats in the model group were significantly lower than those in the normal group; biochemical indicators DA , NE, 5-HT, BDNF were significantly lower than those in the normal group, while CORT was significantly higher than those in the normal group, indicating that the depression model rats were successfully established.
  • the behavioral indexes body weight gain, the percentage of sugar water consumption, the exercise score of the open-box experiment
  • the biochemical indexes DA, NE, 5-HT, BDNF
  • Table 18 shows the results of the order of behavior and biochemical indexes of rats in each administration group. It can be seen from the table that the antidepressant effect of the extract of group D is the best, and the antidepressant effect of the extract of group C is the worst.
  • the extracts of group D, group B, group F and group C can exert antidepressant effect by resisting the hyperfunction of HPA axis, increasing the content of monoamine neurotransmitters and improving neuronal plasticity.
  • the behavioral indexes and biochemical indexes of the extracts in groups D and B were higher than those in group F; the general trend of group D and group B was group D > group B, but there was no significant difference in the content of 5-HT and DA in the cortex, and other indexes There was significant difference between group D and group B; the general trend of group B and group F was group B > group F, but there was no significant difference in the percentage of sugar water consumption, the content of NE in cortex and hippocampus, and the content of 5-HT in hippocampus; the extract of group C Worst antidepressant effect.
  • Bupleurum edulis yielded very little oil, which could not be collected, about 0.2ml/kg; Bran fried Atractylodes had a small amount of oil, 0.95ml/kg; Angelica sinensis yielded more oil, 4.3ml/kg.
  • Angelica sinensis and bran fried Atractylodes can collect volatile oil together. Bupleurum is combined with water extraction because the amount of oil is very small.
  • OBJECTIVE To screen suitable analytical markers in Angelica sinensis and Bran-fried Atractylodes group to characterize the stability of the extract to preparation process.
  • Angelica The screened ligustilide and ferulic acid are not suitable as analytical markers.
  • Ligustilide is a volatile component, which is affected by the collection state of volatile oil, and fluctuates greatly between extraction batches, and the risk of stability of volatile components is high.
  • the content of ferulic acid is low (about 0.1%) and unstable (sensitive to high temperature and light), which can be quantified in the extract, but cannot be accurately quantified due to the interference of Bupleurum in the preparation.
  • Bran fried Atractylodes The selected atractylone and atractylide III components are not suitable for analytical markers.
  • Atractylodes ketone is a volatile component, affected by the collection state of volatile oil, the fluctuation between extraction batches is large, and the risk of stability of volatile components is high.
  • the content of atractylide III is low (about 0.01%), the detectability in the preparation is poor, and the transfer rate from the extract to the preparation is at risk.
  • the glycyrrhizic acid component was used as an analytical marker for the extracts of this group to characterize the process stability of the extract to preparation. Because the properties of glycyrrhizic acid are relatively stable, it is not affected by long-term decoction, as shown in Figure 3, and the proportion of glycyrrhizic acid prescription is low, which is conducive to the batch correspondence between the two groups of extracts.
  • the extraction groups determined after the experimental investigation are: Bupleurum chinensis and Baishao combined extraction to obtain Xiaoyao Chaibai extract; Angelica, bran fried Atractylodes, Zhigancao combined extraction to obtain Xiaoyaodang Baigan extract.
  • the content analysis method of ferulic acid in the Xiaoyaodangbaigan extract containing Angelica sinensis is qualified, and the extract can achieve comprehensive quality control.
  • glycyrrhizic acid composition stability can be used as an analytical marker for this set of extracts to characterize the process stability of extract to formulation.

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Abstract

一种具有缓解精神及抗抑郁功效的中药组合物及其制备方法，所述中药组合物有两种配方：配方1以重量份计为：柴胡10～30、白芍10～30、麸炒白术15～30、当归10～30、炙甘草15～24；配方2以重量份计为：柴胡10～30、白芍10～30、麸炒白术15～30、当归10～30、茯苓10～30份、炙甘草15～24。

Description

一种具有缓解精神及抗抑郁功效的中药组合物及其制备方法 技术领域
本申请属于中药领域，具体涉及但不限于一种具有缓解精神及抗抑郁功效的中药组合物及其制备方法和应用。
背景技术
抑郁症是一种由各种原因引起的以抑郁为主要症状的常见的精神疾病。随着社会的发展，人们生活、工作压力的增大，人们的精神压力也日益增加，导致抑郁症的发病率一直呈上升趋势，其危害性已越来越引起医药卫生界的高度重视。由于抑郁症的诱因很多且发病机制复杂，从而导致抑郁症诊断识别率很低。
目前市场上治疗抑郁症的药物主要以化药为主，抗抑郁效果不甚理想，并且存在价格昂贵，副作用大等问题。而中草药却有毒副作用小、不良反应小以及作用持久等优点，因此从中草药中寻找理想的抗抑郁药物是近年来研究的热点。
逍遥散出自宋代《太平惠民和剂局方》，试验研究以及临床实践证明其有确切的抗抑郁作用。逍遥散由柴胡、当归、白术、白芍、茯苓、薄荷、生姜、甘草八味中药组成，是四逆散衍化而成，其功效为疏肝解郁，健脾和营，是调和肝脾的常用方剂。按君臣佐使分配，其中柴胡、当归、白芍为君药，白术、茯苓、生姜为臣药，薄荷为佐，炙甘草为使。逍遥散八味药理作用不同，其中柴胡主要为疏肝解郁，当归、白芍为养血柔肝，这三味药相互配合，既补肝又和肝，白术、茯苓、生姜三者合用可健脾，薄荷可助柴胡疏散条达，炙甘草可益气补中，调和诸药。
中国专利CN108653405A、CN107625813A公开了一种由6味中药组成的中药组合物，“所述中药组分按质量份数计为：柴胡20～60、当归20～60、麸炒白术20～60、白芍20～60、薄荷8～20、炙甘草8～20；所述制备方法，包括如下步骤：1)在所述组分药材中加入6-10倍重量的60～90％乙醇提取2次，每次1-3h，滤过，合并滤液，浓缩得醇提清膏；2)药渣加入6-10倍重量的水提取2次，每次1-3h，滤过，合并滤液，浓缩得水提清膏，在水提清膏中加入乙醇使含醇量达60～80％，静置24h，取上清液，浓缩至清膏；3)将清膏与醇提清膏合并，混匀后浓缩至稠膏，微波减压干燥，粉碎过80目筛，即得中药提取物”。
中国专利CN101732427A公开了由“柴胡细粉、当归提取物、白芍提取物、白术提取物、茯苓提取物、甘草提取物、薄荷提取物”组成的药物组合物。
中国专利CN102813906A公开了由“柴胡、当归、炒白术、炙甘草、薄荷、干姜”组成的药物组合物。
中国专利CN104644776A公开了由“柴胡、当归、茯苓、白芍、白术、甘草”制得的治疗月经不调的中成药散剂。
中国专利CN108653405A中公开了采用了先合并醇提得醇提清膏，后水提清膏，在水提清膏中加醇再得清膏，并将醇提清膏和第二次获得的清膏合并的制备方法。逍遥散组方中柴胡、当归、白芍、 白术、甘草有挥发性成分或挥发油，对挥发油类通过水蒸气蒸馏法等方法进行提取也被广泛应用到逍遥类产品的制备中。
中国专利CN101732427A公开了将组方中除柴胡之外的其他组分合并水提并保留挥发物，向水提稠膏中加入乙醇过滤两次，将滤液与挥发物及柴胡细粉合并的制备方法。
中国专利公开了CN104189833A公开了：1)将柴胡、当归、炒白术、薄荷四味药材用水蒸气蒸馏法得挥发油，2)使用环糊精处理挥发油得包合物，3)白芍、蜜炙甘草、茯苓和步骤1)提取得到的药渣合并水提，加入挥发油包合物，制得片剂的制备方法。
中国专利公开了CN102908600A公开了：1)将柴胡、生姜、薄荷、当归提取挥发油，2)提取后的药渣与炒白术、茯苓进行水提，浓缩得稠膏，3)白芍、四分之一当量的炙甘草、剩余当量的当归碎成药粉，4)剩余量的炙甘草制成浸膏，5)合并制得药丸的制备方法。
发明概述
以下是对本文详细描述的主题的概述。本概述并非是为了限制权利要求的保护范围。
本申请提供一种具有缓解精神及抗抑郁功效的中药组合物及其制备方法和应用。
本申请的发明人对现有技术的配方进行了细致的研究，运用现代科学技术，研究逍遥散及其类方的抗抑郁作用机制，从逍遥散及其类方中筛选出了治疗抑郁效果更优越的组方，同时为降低成本，简化工艺，优化质量控制，对制备方法做出了新的探索，从而完成了此项发明创造。
本申请具有如下特点：(1)本申请的发明人经过对各种不同组方的研究后，筛选的组方更为简单，成本较低，在大鼠抗抑郁模型中取得了相较于逍遥丸全方(七味，《中国药典》2015年版第1354-1355)及添加了薄荷的类方(六味)(CN108653405A、CN107625813A)更优越的抗抑郁效果；(2)制备过程仅使用水作为溶剂进行提取，制备工艺更简化，更安全可控，成本更低；(3)本申请的发明人采用了分组的提取工艺，避免了其他成分对当归中阿魏酸定量的干扰，同时，分组提取后每一味药都可以进行质量控制，无相互干扰。具体为：柴胡与当归分开，避免柴胡成分对当归中阿魏酸定量的干扰；每组提取物中至少含有1个稳定的指标性成分以表征提取物到制剂过程的工艺稳定性；将含有挥发性成分的麸炒白术与当归划为一组，在提取过程收集芳香水，在浓缩收膏终点将芳香水加入提取物中，使整体提取物与传统应用水煎剂成分接近。
在第一方面，本申请提供一种具有缓解精神及抗抑郁功效的中药组合物，所述中药组合物由柴胡，白芍，麸炒白术，当归，炙甘草以及任选的加入或不加入茯苓组成的重量份中药原料药配方制备而成，其中不加入茯苓的配方为配方1：
柴胡10～30、白芍10～30、麸炒白术15～30、当归10～30、炙甘草15～24。
其中加入茯苓的配方为配方2：
柴胡10～30、白芍10～30、麸炒白术15～30、当归10～30、茯苓10～30、炙甘草15～24。
在第二方面，本申请提供了上述中药组合物的制备方法，所述制备方法包括如下步骤：按组方配比称取药材，加入4～7倍重量的水，提取1-3次，每次1～3h，提取后滤过，合并两次提取的滤液，静置4h以上，取上清液，浓缩得到提取物；以提取物为药物活性成分，必要时加入药用辅料，制备成药物制剂中药组合物。
在第三方面，本申请还提供了一种药物制剂中药组合物(简称中药制剂组合物)，该中药制剂组 合物为口服制剂或注射剂。在一些示例性实施方案中，该中药组合物可以是任何可服用的药物形式：如：片剂、糖衣片剂、薄膜衣片剂、肠溶衣片剂、胶囊剂、硬胶囊剂、软胶囊剂、口服液、口含剂、颗粒剂、冲剂、丸剂、散剂、膏剂、丹剂、混悬剂、粉剂、溶液剂、注射剂、栓剂、软膏剂、硬膏剂、霜剂、喷雾剂、滴剂或贴剂。
本申请通过上述对逍遥散及近似组方抗抑郁机理、组方划裁、制备工艺的研究可知，逍遥散抗抑郁功效随着组方不同、制备工艺不同，其所得发挥抗抑郁功效的有效成分组成有着很大的不同。本申请按照特定药材及配比，采用特定提取的工艺，得到不同的具有抗抑郁功效的组合物。
在阅读并理解了附图和详细描述后，可以明白其他方面。
附图概述
附图用来提供对本申请技术方案的理解，并且构成说明书的一部分，与本申请的实施例一起用于解释本申请的技术方案，并不构成对本申请技术方案的限制。
图1：5味药材合并提取阿魏酸含量分析方法专属性不合格，其中E03为本申请实施例14。
图2：逍遥当白甘提取物中阿魏酸含量分析方法专属性合格。
图3：甘草酸含量变化曲线图。
详述
在第一方面，本申请提供一种具有缓解精神及抗抑郁功效的中药组合物，所述中药组合物由柴胡，白芍，麸炒白术，当归，炙甘草以及任选的加入或不加入茯苓组成的重量份中药原料药配方制备而成，其中不加入茯苓的配方为配方1：
柴胡10～30、白芍10～30、麸炒白术15～30、当归10～30、炙甘草15～24。
其中加入茯苓的配方为配方2：
柴胡10～30、白芍10～30、麸炒白术15～30、当归10～30、茯苓10～30、炙甘草15～24。
在一些示例性实施方案中，按重量份计，所述配方1为：柴胡15～25、白芍15～25、麸炒白术15～25、当归15～25、炙甘草15～20；所述配方2为：柴胡15～25、白芍15～25、麸炒白术15～25、当归15～25、茯苓15～25、炙甘草15～20。
在一些示例性实施方案中，按重量份计，所述配方1为：柴胡20、白芍20、麸炒白术20、当归20、炙甘草16；其中所述配方2为：柴胡20、白芍20、麸炒白术20、当归20、茯苓20、炙甘草16。
在一些示例性实施方案中，所述配方1的中药组合物中包括折干重量逍遥柴白提取物9份，所述逍遥柴白提取物是由柴胡10～30、白芍10～30提取得到的；折干重量逍遥当白甘提取物32份，所述逍遥当白甘提取物是由麸炒白术15～30、当归10～30、炙甘草15～24提取得到的；其中所述逍遥柴白提取物中柴胡皂苷B2折干含量0.63～10.0mg/g、芍药苷折干含量9.4～115.9mg/g；所述逍遥当白甘提取物中甘草酸折干含量1.4～26.1mg/g、阿魏酸折干含量0.13～0.91mg/g、白术内酯III折干含量0.0038～0.30mg/g。
在一些示例性实施方案中，所述逍遥柴白提取物中柴胡皂苷B2折干含量0.94～6.8mg/g、芍药苷折干含量17.6～110.9mg/g；所逍遥当白甘提取物中甘草酸折干含量6.9～26.1mg/g、阿魏酸折干含量0.16～0.79mg/g、白术内酯III折干含量0.0047～0.25mg/g。
本申请所述折干重量，即折算成干燥样品后的重量，也即干燥后的重量。在第二方面，本申请提供了上述中药组合物的制备方法，所述制备方法包括如下步骤：按组方配比称取药材，加入4～7倍重量的水，提取1-3次，每次1～3h，提取后滤过，合并两次提取的滤液，静置4h以上，取上清液，浓缩得到提取物；以提取物为药物活性成分，必要时加入药用辅料，制备成药物制剂中药组合物。
在一些示例性实施方案中，所述制备方法包括如下步骤：按组方配比称取药材，加入6-7倍重量的水，提取2-3h，过滤，药渣加入5-6倍重量的水，提取1h，过滤，合并两次提取的滤液，静置4h以上，取上清液，浓缩，浓缩终点控制在糖度65％～70％，以提取物为药物活性成分，必要时加入药用辅料，制备成药物制剂中药组合物。
在一些示例性实施方案中，所述配方1的中药组合物的制备方法，包括如下步骤：
(1)逍遥柴白提取物的提取：在柴胡、白芍药材中加入4～7倍重量的水，提取1～3次，每次1～3h，提取后滤过，合并两次提取的滤液，静置4h以上，取上清液，浓缩即得；
(2)逍遥当白甘提取物的提取：在当归、麸炒白术、炙甘草药材中加入4～7倍重量的水，提取1～3次，每次1～3h，提取结束收集挥发油和芳香水，提取后提取液过滤，合并两次提取的提取液静置4h以上，取上清液，浓缩，浓缩，加入所收集的挥发油和芳香水，再次浓缩即得；
(3)取逍遥柴白提取物和逍遥当白甘提取物按照配比混合均匀，以逍遥柴白提取物和逍遥当白甘提取物为药物活性成分，必要时加入药用辅料，制备成药物制剂中药组合物。
在一些示例性实施方案中，所述配方1的中药组合物的制备方法，包括如下步骤：
(1)逍遥柴白提取物的提取：在柴胡、白芍药材中加入6倍重量的水，提取2h，过滤，药渣加入5倍重量的水，提取1h，过滤，合并两次提取的滤液，静置4h以上，取上清液，浓缩，浓缩终点密度控制在1.26～1.28，即得；
(2)逍遥当白甘提取物的提取：在当归、麸炒白术、炙甘草药材中加入6倍重量的水，提取2h，过滤，药渣加入5倍重量的水，提取1h，提取结束收集挥发油和芳香水，提取液过滤，合并两次提取的提取液静置4h以上，取上清液，浓缩，浓缩至终点，加入所收集的挥发油和芳香水，再次浓缩至终点，浓缩终点密度控制在1.29～1.31。
(3)取逍遥柴白提取物和逍遥当白甘提取物按照配比混合均匀，以逍遥柴白提取物和逍遥当白甘提取物为药物活性成分，必要时加入药用辅料，制备成药物制剂中药组合物。
本申请的发明人在薄荷和茯苓减方后，将剩余5味药材合并水提时进一步发现，当归中阿魏酸成分的定量受到柴胡某些成分的干扰，专属性不合格，如图1所示。考虑到当归中阿魏酸成分可定量且相对稳定，发明人进行了良好的提取工艺设计，通过分组提取的方式，避免了其他成分对当归中阿魏酸定量的干扰，如图2。分组提取，以提取物入药，不仅药效没有降低，其质量得到有效控制。
柴胡药材里能测到的皂苷成分是柴胡皂苷A和柴胡皂苷D，提取过程中发生了水解，柴胡皂苷D转化为B2，在提取物中可检测到柴胡皂苷B2且折干含量最高。
在第三方面，本申请还提供了一种药物制剂中药组合物(简称中药制剂组合物)，该中药制剂组合物可以是口服制剂或注射剂。
其中，所述口服制剂选自胶囊剂、片剂、滴丸、颗粒剂、浓缩丸、口服液和合剂中的一种。
其中，所述注射剂选自注射液、冻干粉针剂和水针剂中的一种。
在一些示例性实施方案中，本申请的中药制剂组合物是任何可用的药物形式：如：片剂、糖衣片剂、薄膜衣片剂、肠溶衣片剂、胶囊剂、硬胶囊剂、软胶囊剂、口服液、口含剂、颗粒剂、冲剂、丸剂、散剂、膏剂、丹剂、混悬剂、粉剂、溶液剂、注射剂、栓剂、软膏剂、硬膏剂、霜剂、喷雾剂、滴剂或贴剂。
在一些示例性实施方案中，本申请的中药制剂组合物是单位剂量的药物制剂形式。
本申请的中药制剂组合物，在制成药剂时，单位剂量的药剂可含有本申请的中药组合物0.1-1000mg，其余为药学上可接受的辅料。药学上可接受的辅料以重量计可以是制剂总重量的0.01-99.99％。
本申请的中药制剂在使用时根据病人的情况确定用法用量，如一日1-3次、一次1-20片等。
本申请的中药制剂组合物，其口服给药的制剂可含有常用的药用辅料，诸如但不限于粘合剂、填充剂、稀释剂、压片剂、润滑剂、崩解剂、着色剂、调味剂和湿润剂，必要时可对片剂进行包衣。
适用的填充剂包括纤维素、甘露糖醇、乳糖和其它类似的填充剂。适宜的崩解剂包括淀粉、聚乙烯吡咯烷酮和淀粉衍生物，例如羟基乙酸淀粉钠。适宜的润滑剂包括，例如硬脂酸镁。适宜的药物可接受的湿润剂包括十二烷基硫酸钠。
本申请的中药制剂组合物可通过混合，填充，压片等常用方法制备固体口服组合物。进行反复混合可使活性物质分布在整个使用大量填充剂的那些组合物中。
口服液体制剂的形式例如可以是水性或油性悬浮液、溶液、乳剂、糖浆剂或酏剂，或者可以是一种在使用前可用水或其它适宜的载体复配的干燥产品。这种液体制剂可含有常规的添加剂，诸如悬浮剂，例如山梨醇、糖浆、甲基纤维素、明胶、羟乙基纤维素、羧甲基纤维素、硬脂酸铝凝胶或氢化食用脂肪，乳化剂，例如卵磷脂、脱水山梨醇一油酸酯或阿拉伯胶；非水性载体(它们可以包括食用油)，例如杏仁油、分馏椰子油、诸如甘油的酯的油性酯、丙二醇或乙醇；防腐剂，例如对羟基苯甲酯或对羟基苯甲酸丙酯或山梨酸，并且如果需要，可含有常规的香味剂或着色剂。
对于注射剂，制备的液体单位剂型含有本申请的活性物质和无菌载体。根据载体和浓度，可以将此活性物质悬浮或者溶解。溶液的制备通常是通过将活性物质溶解在一种载体中，再将其装入一种适宜的小瓶或安瓿前过滤消毒，然后密封。辅料例如一种局部麻醉剂、防腐剂和缓冲剂也可以溶解在这种载体中。为了提高其稳定性，可在装入小瓶以后将这种组合物冰冻，并在真空下将水除去。
下面结合具体实施例对本申请作进一步的说明。
实施例1
按组方配比称取柴胡20kg、白芍20kg，加入6倍重量的水，提取2h，过滤，药渣加入5倍重量的水，提取1h，过滤，合并两次提取的滤液，常温条件下静置4h以上，取上清液，浓缩至相对密度1.27，得逍遥柴白提取物。其中柴胡皂苷B2折干含量2.7mg/g、芍药苷折干含量42.1mg/g。
实施例2
按组方配比称取柴胡20kg、白芍20kg，加入7倍重量的水，提取3h，过滤，药渣加入4倍重量的水，提取1h，过滤，合并两次提取的滤液，常温条件下静置4h以上，取上清液，浓缩至相对密度1.27，得逍遥柴白提取物。其中柴胡皂苷B2折干含量2.9mg/g、芍药苷折干含量40.8mg/g。
实施例3
按组方配比称取柴胡15kg、白芍25kg，加入6倍重量的水，提取2h，过滤，药渣加入5倍重量 的水，提取1h，过滤，合并两次提取的滤液，常温条件下静置4h以上，取上清液，浓缩至相对密度1.27，得逍遥柴白提取物。其中柴胡皂苷B2折干含量2.1mg/g、芍药苷折干含量54.2mg/g。
实施例4
按组方配比称取柴胡25kg、白芍15kg，加入7倍重量的水，提取3h，过滤，药渣加入4倍重量的水，提取1h，过滤，合并两次提取的滤液，常温条件下静置4h以上，取上清液，浓缩至相对密度1.27，得逍遥柴白提取物。其中柴胡皂苷B2折干含量3.1mg/g、芍药苷折干含量30.9mg/g。
实施例5
按组方配比称取柴胡30kg、白芍10kg，加入6倍重量的水，提取2h，过滤，药渣加入5倍重量的水，提取1h，过滤，合并两次提取的滤液，常温条件下静置4h以上，取上清液，浓缩至相对密度1.27，得逍遥柴白提取物。其中柴胡皂苷B折干含量23.7mg/g、芍药苷折干含量22.2mg/g。
实施例6
按组方配比称取柴胡10kg、白芍30kg，加入7倍重量的水，提取3h，过滤，药渣加入4倍重量的水，提取1h，过滤，合并两次提取的滤液，常温条件下静置4h以上，取上清液，浓缩至相对密度1.27，得逍遥柴白提取物。其中柴胡皂苷B折干含量21.4mg/g、芍药苷折干含量65.70mg/g。
实施例7
按组方配比称取当归20kg、麸炒白术20kg、炙甘草16kg，加入6倍重量的水，提取2h，过滤，药渣加入5倍重量的水，提取1h，提取结束收集挥发油和芳香水，提取液过滤，合并两次提取的提取液，常温条件下静置4h以上，取上清液浓缩至相对密度1.30，加入所收集的挥发油和芳香水，继续浓缩至相对密度1.30，得逍遥当白甘提取物。其中甘草酸折干含量15.7mg/g、阿魏酸折干含量0.50mg/g、白术内酯III折干含量0.0.0078mg/g。
实施例8
按组方配比称取当归20kg、麸炒白术20kg、炙甘草16kg，加入7倍重量的水，提取3h，过滤，药渣加入4倍重量的水，提取1h，提取结束收集挥发油和芳香水，提取液过滤，合并两次提取的提取液，常温条件下静置4h以上，取上清液浓缩至相对密度1.30，加入所收集的挥发油和芳香水，继续浓缩至相对密度1.30，得逍遥当白甘提取物。其中甘草酸折干含量15.4mg/g、阿魏酸折干含量0.48mg/g、白术内酯III折干含量0.0073mg/g
实施例9
按组方配比称取当归25kg、麸炒白术15kg、炙甘草15kg，加入6倍重量的水，提取2h，过滤，药渣加入5倍重量的水，提取1h，提取结束收集挥发油和芳香水，提取液过滤，合并两次提取的提取液，常温条件下静置4h以上，取上清液浓缩至相对密度1.30，加入所收集的挥发油和芳香水，继续浓缩至相对密度1.30，得逍遥当白甘提取物。其中甘草酸折干含量11.9mg/g、阿魏酸折干含量0.62mg/g、白术内酯III折干含量0.0058mg/g。
实施例10
按组方配比称取当归25kg、麸炒白术15kg、炙甘草20kg，加入7倍重量的水，提取3h，过滤，药渣加入4倍重量的水，提取1h，提取结束收集挥发油和芳香水，提取液过滤，合并两次提取的提取液，常温条件下静置4h以上，取上清液浓缩至相对密度1.30，加入所收集的挥发油和芳香水，继续浓 缩至相对密度1.30，得逍遥当白甘提取物。甘草酸折干含量18.8mg/g、阿魏酸折干含量0.59mg/g、白术内酯III折干含量0.0055mg/g。
实施例11
按组方配比称取当归15kg、麸炒白术25kg、炙甘草18kg，加入6倍重量的水，提取2h，过滤，药渣加入5倍重量的水，提取1h，提取结束收集挥发油和芳香水，提取液过滤，合并两次提取的提取液，常温条件下静置4h以上，取上清液浓缩至相对密度1.30，加入所收集的挥发油和芳香水，继续浓缩至相对密度1.30，得逍遥当白甘提取物。其中甘草酸折干含量16.9mg/g、阿魏酸折干含量0.36mg/g、白术内酯III折干含量0.0092mg/g。
实施例12
按组方配比称取当归30kg、麸炒白术15kg、炙甘草24kg，加入7倍重量的水，提取3h，过滤，药渣加入4倍重量的水，提取1h，提取结束收集挥发油和芳香水，提取液过滤，合并两次提取的提取液，常温条件下静置4h以上，取上清液浓缩至相对密度1.30，加入所收集的挥发油和芳香水，继续浓缩至相对密度1.30，得逍遥当白甘提取物。甘草酸折干含量21.4mg/g、阿魏酸折干含量0.67mg/g、白术内酯III折干含量0.0047mg/g。
实施例13
按组方配比称取当归10kg、麸炒白术30kg、炙甘草15kg，加入7倍重量的水，提取3h，过滤，药渣加入4倍重量的水，提取1h，提取结束收集挥发油和芳香水，提取液过滤，合并两次提取的提取液，常温条件下静置4h以上，取上清液浓缩至相对密度1.30，加入所收集的挥发油和芳香水，继续浓缩至相对密度1.30，得逍遥当白甘提取物。甘草酸折干含量7.4mg/g、阿魏酸折干含量0.23mg/g、白术内酯III折干含量0.0154mg/g。
实施例14
取实施例1逍遥柴白提取物9kg(折干重量)、实施例7逍遥当白甘提取物32kg(折干重量)混匀，即得本申请的中药组合物。
实施例15
取实施例1-6任意一项逍遥柴白提取物、实施例7-13任意一项逍遥当白甘提取物按照折干重量比9:32混合均匀即本申请的中药组合物。
实施例16
柴胡20kg、白芍20kg、麸炒白术20kg、当归20kg、炙甘草16kg。按组方配比称取药材，加入6倍重量的水，提取2h，过滤，药渣加入5倍重量的水，提取1h，过滤，合并两次提取的滤液，常温条件下静置4h以上，取上清液，浓缩终点控制在糖度65％～70％，得到中药提取物。
实施例17
柴胡10kg、白芍10kg、麸炒白术15kg、当归10kg、炙甘草15kg。按照实施例16制备。按组方配比称取药材，加入6倍重量的水，提取2h，过滤，药渣加入5倍重量的水，提取1h，过滤，合并两次提取的滤液，常温条件下静置4h以上，取上清液，浓缩终点控制在糖度68％～72％，得到中药提取物。
实施例18
柴胡30kg、白芍30kg、麸炒白术30kg、当归30kg、炙甘草24kg。按组方配比称取药材，加入6倍重量的水，提取3h，过滤，药渣加入4倍重量的水，提取1h，过滤，合并两次提取的滤液，常温条件下静置4h以上，取上清液，浓缩至糖度65％～70％，得到提取物。
实施例19
柴胡15kg、白芍15kg、麸炒白术15kg、当归15kg、炙甘草15kg。按组方配比称取药材，加入6倍重量的水，提取3h，过滤，药渣加入4倍重量的水，提取1h，过滤，合并两次提取的滤液，常温条件下静置4h以上，取上清液，浓缩至糖度65％～70％，得到提取物。
实施例20
柴胡25kg、白芍25kg、麸炒白术25kg、当归25kg、炙甘草20kg。按组方配比称取药材，加入6倍重量的水，提取3h，过滤，药渣加入4倍重量的水，提取1h，过滤，合并两次提取的滤液，常温条件下静置4h以上，取上清液，浓缩至糖度65％～70％，得到提取物。
实施例21
实施例16-20中的组方配比称取药材，加入7倍重量的水，提取3h，过滤，药渣加入5倍重量的水，提取1h，过滤，合并两次提取的滤液，常温条件下静置4h以上，取上清液，浓缩至糖度65％～70％，得到提取物。
实施例22
柴胡15kg、白芍15kg、麸炒白术15kg、当归15kg、炙甘草15kg。按组方配比称取药材，加入7倍重量的水，提取3h，过滤，药渣加入4倍重量的水，提取1h，过滤，合并两次提取的滤液，常温条件下静置4h以上，取上清液，浓缩至糖度65％～70％，得到提取物。
实施例23
柴胡25kg、白芍25kg、麸炒白术25kg、当归25kg、炙甘草20kg。按组方配比称取药材，加入7倍重量的水，提取3h，过滤，药渣加入4倍重量的水，提取1h，过滤，合并两次提取的滤液，常温条件下静置4h以上，取上清液，浓缩至糖度65％～70％，得到提取物。
实施例24
柴胡10kg、白芍10kg、麸炒白术15kg、当归10kg、茯苓8kg、炙甘15kg。按组方配比称取药材，加入6倍重量的水，提取2h，过滤，药渣加入5倍重量的水，提取1h，过滤，合并两次提取的滤液，常温条件下静置4h以上，取上清液，浓缩终点控制在糖度65％～70％，得到中药提取物。
实施例25
柴胡30kg、白芍30kg、麸炒白术30kg、当归30kg、茯苓24kg、炙甘草24kg。按组方配比称取药材，加入7倍重量的水，提取3h，过滤，药渣加入4倍重量的水，提取1h，过滤，合并两次提取的滤液，常温条件下静置4h以上，取上清液，浓缩至糖度65％～70％，得到提取物。
实施例26
柴胡15kg、白芍15kg、麸炒白术15kg、当归15kg、茯苓15kg、炙甘草15kg。按组方配比称取药材，加入6倍重量的水，提取2h，过滤，药渣加入5倍重量的水，提取1h，过滤，合并两次提取的滤液，常温条件下静置4h以上，取上清液，浓缩至糖度65％～70％，得到提取物。
实施例27
柴胡25kg、白芍25kg、麸炒白术25kg、当归25kg、茯苓25kg、炙甘草20kg。按组方配比称取药材，加入7倍重量的水，提取3h，过滤，药渣加入4倍重量的水，提取1h，过滤，合并两次提取的滤液，常温条件下静置4h以上，取上清液，浓缩至糖度65％～70％，得到提取物。
实施例28
柴胡20kg、白芍20kg、麸炒白术20kg、当归20kg、茯苓20kg、炙甘草16kg。按组方配比称取药材，加入7倍重量的水，提取2h，过滤，药渣加入6倍重量的水，提取1h，过滤，合并两次提取的滤液，静置4h以上，取上清液，浓缩至糖度65％～70％，得提取物。
实施例29：含量测定
下述含量测定中所述的折干含量＝提取物含量/提取物干燥残留，提取物干燥残留等同于固含量，检测方法参照欧洲药典<2.8.16>。
(1)柴胡皂苷B2含量测定
色谱条件：Waters UPLC色谱仪，色谱柱：BEH C18(2.1mm*100mm，1.7μm)色谱柱，流动相为乙腈(A)-水(B)，按下表进行梯度洗脱，检测波长254nm，柱温30℃。
时间(min) 流速(ml/min) 流动相A(乙腈) 流动相B(水)
0 0.3 10 90
6 0.3 38 62
11 0.3 40 60
14 0.3 100 0
16 0.3 100 0
18 0.3 10 90
20 0.3 10 90
对照品溶液制备：
精密称取柴胡皂苷B标准品27.5mg，置于50ml量瓶中，加甲醇15ml使溶解，定容，精密移取5ml，置于50ml量瓶中，用甲醇定容，混匀即得。
供试品溶液制备：
精密称取本品0.2g，置于50ml离心管中，加60％甲醇-0.5mol/L NaOH溶液20ml，超声40min，离心10min，精密量取10ml上样于PSZG柱(先用10ml甲醇和10ml纯化水活化)，用水洗脱至洗脱液中性，再用25ml甲醇洗脱并收集洗脱液，50℃减压浓缩至干，用甲醇复溶至5ml容量瓶中，过0.22μm有机滤膜即得。
60％甲醇-0.5mol/LNaOH溶液：取氢氧化钠2g，加水使溶解成100ml，即得0.5mol/L NaOH溶液，取甲醇60ml，加0.5mol/L NaOH溶液使成100ml，混合均匀，即得。(可根据实际需求，按比例调整各试药、试剂加入量)
测定法
分别精密吸取对照品溶液、供试品溶液各2μl注入超高效液相色谱仪，测定，用外标法计算柴胡皂苷B2折干含量。
计算公式：
Figure PCTCN2021109326-appb-000001
Figure PCTCN2021109326-appb-000002
柴胡皂苷B2折干含量为0.63～10.0mg/g
注：Content(柴胡皂苷B2)：柴胡皂苷B2含量，单位mg/g；
A 供试品：供试品峰面积；
C 对照品：对照品溶液浓度，单位mg/ml；
10：稀释倍数；
H：对照品纯度；
A 对照品：对照品峰面积；
m：供试品称样量，单位g；
Content(柴胡皂苷B2)折干：柴胡皂苷B2折干含量，单位mg/g；
D：干燥残留，单位％。
方法学验证数据：
进样稳定性：对照品溶液：36h；供试品溶液：36h
样品含量结果平行性评价：RAD％≤3.0％
(2)芍药苷含量测定
色谱条件：
Waters UPLC色谱仪，色谱柱：HSS T3(2.1mm*100mm，1.8μm)，以乙腈为流动相A，以0.05％磷酸水溶液为流动相B，按下表进行梯度洗脱；柱温为30℃，检测波长：230nm，进样量2μl。
0.05％磷酸水溶液：取磷酸0.5ml，加超纯水使成1000ml，即得。(可根据实际需求，按比例调整各试剂加入量)
时间(min) 流速(ml/min) 流动相A(乙腈) 流动相B(0.05％磷酸水溶液)
0 0.3 8 92
16 0.3 10 90
18 0.3 100 0
19 0.3 100 0
21 0.3 8 92
22 0.3 8 92
对照品溶液制备：
精密称取芍药苷标准品6mg，置于50ml量瓶中，加甲醇15ml使溶解，用纯化水稀释至刻度，混匀即得。
供试品溶液制备：
精密称取本品0.1g，置于25ml量瓶中，加30％甲醇溶液适量，超声20min，充分溶解，用30％甲醇溶液定容，过0.22μm有机滤膜即得。
30％甲醇溶液：取甲醇30ml，加水使成100ml，混合均匀，即得。(可根据实际需求，按比例调 整各试剂加入量)
测定法
分别精密吸取对照品溶液、供试品溶液各2μl注入超高效液相色谱仪，测定，用外标法计算芍药苷折干含量。
计算公式：
Figure PCTCN2021109326-appb-000003
Figure PCTCN2021109326-appb-000004
芍药苷折干含量为9.4～115.9mg/g
注：Content(芍药苷)：芍药苷折干含量，单位mg/g；
A 供试品：供试品峰面积；
C 对照品：对照品溶液浓度，单位mg/ml；
25：供试品溶液体积，单位ml；
H：对照品纯度；
A 对照品：对照品峰面积；
m：供试品称样量，单位g；
Content(芍药苷)折干：芍药苷折干含量，单位mg/g；
D：干燥残留，单位％。
方法学验证数据：
进样稳定性：对照品溶液：24h；供试品溶液：24h
(3)甘草酸含量测定
色谱条件：
Waters UPLC色谱仪，色谱柱：HSS T3(2.1mm*100mm，1.8μm)，以乙腈为流动相A，以0.05％磷酸水溶液为流动相B，按下表规定进行梯度洗脱；柱温为30℃，检测波长230nm。
0.05％磷酸水溶液：取磷酸0.5ml，加超纯水使成1000ml，即得。(可根据实际需求，按比例调整各试剂加入量)
时间(min) 流速(ml/min) 流动相A(乙腈) 流动相B(0.05％磷酸水溶液)
0 0.3 5 95
5 0.4 20 80
16 0.4 55 45
17 0.4 100 0
18 0.4 100 0
19 0.3 5 95
21 0.3 5 95
对照品溶液：
精密称取甘草酸铵标准品6mg，置于50ml量瓶中，加甲醇15ml使溶解，用纯化水稀释至刻度， 精密移取5ml，置于25ml量瓶中，用30％甲醇溶液定容，混匀即得。
供试品溶液：
精密称取本品0.15g，置于25ml量瓶中，加30％甲醇溶液约15ml，超声20min，充分溶解，用30％甲醇溶液定容，过0.22μm有机滤膜即得。
30％甲醇溶液：取甲醇30ml，加水使成100ml，混合均匀，即得。(可根据实际需求，按比例调整各试剂加入量)
测定法
分别精密吸取对照品溶液、供试品溶液各2μl注入超高效液相色谱仪，测定，用外标法计算甘草酸折干含量。
计算公式：
Figure PCTCN2021109326-appb-000005
Figure PCTCN2021109326-appb-000006
甘草酸折干含量为1.4～26.1mg/g
注：Content(甘草酸)：甘草酸折干含量，单位mg/g；
A 供试品：供试品峰面积；
C 对照品：对照品溶液浓度，单位mg/ml；
25：供试品溶液体积，单位ml；
H：对照品纯度；
A 对照品：对照品峰面积；
m：供试品称样量，单位g；
Content(甘草酸)折干：甘草酸折干含量，单位mg/g；
D：干燥残留，单位％。
方法学验证数据：
进样稳定性：对照品溶液：24h；供试品溶液：24h
(4)阿魏酸
色谱条件：
Waters UPLC色谱仪，色谱柱：HSS T3(2.1mm*100mm，1.8μm)，以乙腈为流动相A，以0.05％磷酸水溶液为流动相B，按下表进行梯度洗脱；柱温为30℃，样品温度20℃，检测波长：323nm，进样量2μl。
0.05％磷酸水溶液：取磷酸0.5ml，加超纯水使成1000ml，即得。(可根据实际需求，按比例调整各试剂加入量)
时间(min) 流速(ml/min) 流动相A(乙腈) 流动相B(0.05％磷酸水溶液)
0 0.4 5 95
3 0.4 11 89
12 0.4 12 88
14 0.4 20 80
16 0.4 30 70
17 0.4 100 0
19 0.4 5 95
20 0.4 5 95
对照品溶液制备：
精密称取阿魏酸标准品6mg，置于50ml量瓶中，加甲醇15ml使溶解，用纯化水稀释至刻度，精密移取1ml，置于50ml量瓶中，用30％甲醇溶液定容，混匀即得。
供试品溶液制备：
精密称取本品0.15g，置于25ml量瓶中，加30％甲醇溶液约15ml，超声20min，充分溶解，用30％甲醇溶液定容，过0.22μm有机滤膜即得。
30％甲醇溶液：取甲醇30ml，加水使成100ml，混合均匀，即得。(可根据实际需求，按比例调整各试剂加入量)
测定法：
分别精密吸取对照品溶液、供试品溶液各2μl注入超高效液相色谱仪，测定，用外标法计算阿魏酸折干含量。
计算公式：
Figure PCTCN2021109326-appb-000007
Figure PCTCN2021109326-appb-000008
阿魏酸折干含量为0.13～0.91mg/g
注：Content(阿魏酸)：阿魏酸折干含量，单位mg/g；
A 供试品：供试品峰面积；
C 对照品：对照品溶液浓度，单位mg/ml；
25：供试品溶液体积，单位ml；
H：对照品纯度；
A 对照品：对照品峰面积；
m：供试品称样量，单位g；
Content(阿魏酸)折干：阿魏酸折干含量，单位mg/g；
D：干燥残留，单位％。
方法学验证数据：
进样稳定性：对照品溶液：36h；供试品溶液：36h
(5)白术内酯III
色谱条件：
Waters UPLC色谱仪，色谱柱：HSS T3(2.1mm*100mm，1.8μm)，以乙腈为流动相A，以0.05％ 磷酸水溶液为流动相B，按下表进行梯度洗脱；柱温为30℃，样品温度20℃，检测波长：220nm，进样量2μl。
0.05％磷酸水溶液：取磷酸0.5ml，加超纯水使成1000ml，即得。(可根据实际需求，按比例调整各试剂加入量)
时间(min) 流速(ml/min) 流动相A(乙腈) 流动相B(0.05％磷酸水溶液)
0 0.3 10 90
12 0.3 12 88
14 0.3 12 88
22 0.3 28 72
34 0.3 45 55
36 0.3 80 20
39 0.3 90 10
41 0.3 90 10
42 0.3 10 90
44 0.3 10 90
对照品溶液的制备：
精密称取白术内酯III标准品6mg，置于50ml量瓶中，加甲醇15ml使溶解，用纯化水稀释至刻度，精密移取5ml，置于50ml量瓶中，用30％甲醇溶液定容，混匀即得。
供试品溶液的制备：
精密称取本品0.5g，置于10ml量瓶中，加30％甲醇溶液约7ml，超声30min，充分溶解，用30％甲醇溶液定容，离心，过0.22μm有机滤膜即得。
30％甲醇溶液：取甲醇30ml，加水使成100ml，混合均匀，即得。(可根据实际需求，按比例调整各试剂加入量)
测定法：
分别精密吸取对照品溶液、供试品溶液各2μl注入超高效液相色谱仪，测定，用外标法计算白术内酯III折干含量。
计算公式：
Figure PCTCN2021109326-appb-000009
Figure PCTCN2021109326-appb-000010
白术内酯III折干含量为0.0038～0.30mg/g
注：Content(白术内酯III)：白术内酯Ⅲ含量，单位mg/g；
A 供试品：供试品峰面积；
C 对照品：对照品溶液浓度，单位mg/ml；
10：供试品溶液体积，单位ml；
H：对照品纯度；
A 对照品：对照品峰面积；
m：供试品称样量，单位g；
Content(白术内酯III)折干：白术内酯III折干含量，单位mg/g；
D：干燥残留，单位％。
方法学验证数据：
进样稳定性：对照品溶液：48h；供试品溶液：48h
样品含量结果平行性评价：RAD％≤8.5％
实施例30抗抑郁药效实验
试验例一
慢性不可预知温和刺激(Chronic unpredictable mild stress，CUMS)结合孤养抑郁大鼠模型是与临床抑郁症最接近的动物模型，它是指在造模过程中将大鼠暴露于持续的温和不可预见的刺激条件之下，并结合孤养诱导大鼠产生与抑郁症患者类似的许多行为学异常症状。其中体重变化、糖水消耗百分比、敞箱实验运动得分是该抑郁模型的主要行为学评价指标，能反映临床抑郁症患者的食欲不振、快感缺失、自主活动能力及对新鲜环境的好奇程度降低等症状。
抑郁症发病机制复杂，目前被广泛认可且研究较深入的假说包括单胺类神经递质假说、下丘脑-垂体-肾上腺(HPA)轴功能亢进假说、神经可塑性假说。其中单胺类神经递质假说作为抑郁症最经典的一种假说，认为抑郁症的发生主要是由于大脑中枢神经系统内单胺类神经递质如5-羟色胺(5-HT)、多巴胺(DA)和去甲肾上腺素(NE)等缺乏密切相关，这也是目前临床大部分抗抑郁药的作用靶点；下丘脑-垂体-肾上腺(HPA)轴功能亢进可导致机体皮质酮(CORT)含量异常升高，与其受体结合后产生神经毒性，造成海马区神经元损伤，从而引起抑郁；神经可塑性假说认为脑源性神经营养因子(BDNF)参与调控神经细胞的生存、生长、分化和凋亡，可对抗应激状态下神经元的损伤，促进神经元的修复与再生，提高神经元的可塑性，从而发挥抗抑郁作用。因此，本实验采用CUMS结合孤养抑郁大鼠模型，以CUMS结合孤养抑郁模型大鼠的行为学及生化指标(CORT、DA、NE、5-HT、BDNF)含量为抗抑郁作用药效学指标，比较加味逍遥丸减方前后的抗抑郁作用药效学。
1实验动物与材料
1.1实验动物
SPF级雄性Sprague Dawley(SD)大鼠130只[购于中国食品药品检定研究院，许可证号：SCXK(京)2017-0005]，体重180～200g，适应性饲养及实验地点均为天津中医药大学实验动物中心。饲养条件：(22±2)℃，湿度(50±10)％，自然光照，自由摄食和饮水。实验动物的饲养和操作规程均遵守天津中医药大学实验动物饲养和使用的相关规定。
1.2药品与试剂
A组(加味逍遥方)、C组(逍遥方)、D组(本申请实施例16)、E组(本申请实施例28)、F组提取物(参考中国专利CN108653405A制备)按照表1中配方制备均由天士力医药集团股份有限公司提供；盐酸氟西汀(Flu)产于礼来苏州制药有限公司；血浆皮质酮(CORT)ELISA试剂盒、神经营养因子(BDNF)ELISA试剂盒、5-羟色胺(5-HT)ELISA试剂盒、多巴胺(DA)ELISA试剂盒、 去甲肾上腺素(NE)ELISA试剂盒均购自美国R&D Systems；生理盐水购自石家庄四药有限公司。
1.3实验设备与仪器
敞箱(100cm×100cm×50cm，自制)，LTD0114子弹头警灯(浙江安华安全设备有限公司)，G6805-1治疗仪(中国青岛鑫升实业有限公司)，木质夹子，TD21001电子天平(天津市天马仪器厂)，IKA T18型超声组织破碎仪(宁波新芝生物科技有限公司)，3K15型冷冻高速离心机(美国Sigma公司)，Infinite M200Pro型多功能酶标仪(瑞士TECAN公司)，-80℃低温冰箱(美国Thermo科技公司)。
2实验方法
2.1药物溶液配制
2.1.1氟西汀混悬液的配制
取盐酸氟西汀胶囊(20mg/粒)，除去胶囊壳，加入0.5％CMC-Na溶液研磨配制成1mg/mL的氟西汀混悬液。
2.1.2各组药液的配制
A组、C组、D组、E组、F组提取物每天临床用量分别为5.21g、3.21g、3.26g、3.29g、2.55g，按15倍临床等效剂量折合成大鼠的剂量分别为7.0305g/kg、4.3380g/kg、4.4040g/kg、4.4415g/kg、3.4425g/kg，具体见表1。
表1 大鼠灌胃剂量折算表
Figure PCTCN2021109326-appb-000011
Figure PCTCN2021109326-appb-000012
出膏率(％)＝提取物重量/药材重量×100％
A组药、B组药、C组药提取方法：
各自按组方配比称取所有药材，加入6倍重量的水，提取2h，过滤，药渣加入5倍重量的水，提取1h，过滤，合并两次提取的滤液，常温条件下静置4h以上，取上清液，浓缩终点控制在糖度65％～70％，得到中药提取物。
2.2CUMS结合孤养抑郁模型建立
将130只SD大鼠适应性饲养7天后，在安静环境下对大鼠进行敞箱实验，根据水平运动和垂直运动得分相近的原则筛选大鼠，剔除得分过高或过低的大鼠。将筛选出大鼠随机分成正常组、模型组、Flu组、A组、C组、D组、E组、F组，每组13只。
正常组大鼠每笼4只常规饲养，自由饮水摄食，不接受任何其他额外刺激；其余各组大鼠均每只单笼饲养，每天随机接受以下2种不同应激刺激且相同刺激不可连续出现，从而使大鼠不能预料何种刺激，避免其产生适应性。CUMS刺激因子包括：束缚2h，冰水高台2h，噪音刺激(110dB，30min)，足底电击(电流4mA，50次，每次间隔5s)，光照刺激(100 000lx，30min)，昼夜颠倒24h，潮湿垫料12h，夹尾(距离大鼠尾尖1cm左右，2min)。持续造模，每周测量各组大鼠体重增长量、糖水消耗百分比及敞箱实验水平运动与垂直运动得分。
2.3药效学测定指标
2.3.1行为学实验
(1)体重变化
实验第0、7、14、21、28、35、42、49、56、63天分别对大鼠进行体重测量，记录各组大鼠体重，分析造模过程中各组大鼠每周体重变化及造模前后体重增长量(％)结果。公式如下：
Figure PCTCN2021109326-appb-000013
(2)糖水消耗实验
实验第0、7、14、21、28、35、42、49、56、63天对各组大鼠进行糖水消耗实验。实验前3天进行糖水适应，在笼子上放置两瓶1％浓度蔗糖溶液24h，然后放置1％蔗糖溶液一瓶、纯水一瓶24h，上下午调换水瓶位置，第3天禁食禁水24h。正式实验各组大鼠分别放置1％蔗糖溶液一瓶、纯水一瓶，上下午调换水瓶位置，记录各组大鼠24h内蔗糖溶液量(糖水消耗量)及纯水消耗量，计算糖水消耗百分比(％)。公式如下：
Figure PCTCN2021109326-appb-000014
(3)敞箱实验
实验第0、7、14、21、28、35、42、49、56、63天对各组大鼠进行敞箱实验。实验所用敞箱大小为100cm×100cm×50cm(长×宽×高)，内侧面黑色，将底面用白线划分成100(10cm×10cm)个大小相同小方格。将每只大鼠依次放在中心固定的方格内，排除人为干扰，记录大鼠3min内活动路线。以实验大鼠穿越底面小方格的数量作为水平运动得分，大鼠每穿越1格(3爪以上跨入)均计为水平运动1分，若实验大鼠沿直线行走，则以每10cm计为1分。以实验大鼠直立次数作为垂直运动得分，大鼠双前肢离开敞箱底面为标志，无论大鼠站立多久，均以大鼠放下双前肢计为垂直运动1分。每只大鼠测试完毕后，彻底清理敞箱，再进行下一只大鼠的测试。记录每只大鼠的水平运动得分及垂直运动得分，分析各组大鼠水平运动得分及垂直运动得分差异。
2.3.2生化指标检测
(1)取材
行为学实验后，大鼠眼眶取血约1.5mL，置于肝素浸润的离心管中，8000r·min -1，4℃，离心10min，取上清液，置于-80℃低温冰箱中保存，用于大鼠血浆中皮质酮(CORT)水平的测定。
眼眶取血后，脱颈处死，在冰面上迅速剥离出皮层和海马，称重，置冻存管中在液氮中迅速冷冻，然后转移至-80℃低温冰箱中保存，用于大鼠皮层与海马中神经生长营养因子(BDNF)、5-羟色胺(5-HT)、多巴胺(DA)和去甲肾上腺素(NE)含量的测定。
2.3.3.2CORT、BDNF、5-HT、DA、NE测定及计算
采用酶联免疫吸附测定(Enzyme linked immunosorbent assay，ELISA)对各组大鼠血浆中CORT含量及皮层与海马中BDNF、5-HT、DA和NE含量进行测定，在450nm波长下依序测量各孔的吸光度(OD值)。以标准物的含量为横坐标，OD值为纵坐标，绘制标准曲线并得回归方程，将各组大鼠血浆中CORT及皮层与海马中BDNF、5-HT、DA和NE的OD值代入回归方程，计算各组大鼠血浆中CORT及皮层与海马中BDNF、5-HT、DA和NE的含量。
2.4.实验数据处理及统计学分析
实验数据以
Figure PCTCN2021109326-appb-000015
表示，采用SPSS 22.0统计学软件分析，大鼠血浆中CORT含量及皮层与海马中BDNF、5-HT、DA、NE含量采用单因素方差分析(One-way ANOVA)进行组间差异分析。
3实验结果与分析
3.1体重变化结果
各实验组大鼠随着造模过程可见不同程度的体重增加，结果见表2，模型组大鼠体重增长量较正常组显著下降(P<0.01)，表明CUMS结合孤养造模方法影响大鼠的食欲导致体重增长缓慢，模拟了抑郁症患者食欲减退、体重减轻症状。
各给药组大鼠体重增长量较模型组显著升高(P<0.01)，表明各给药组均可抵抗CUMS结合孤养抑郁模型大鼠体重降低，从而改善CUMS结合孤养抑郁模型大鼠食欲减退、体重减轻症状。由表可知：总体上各给药组大鼠体重增长量大小关系依次为Flu组>D组>F组>E组>C组>A组，表明D组提取 物改善CUMS结合孤养抑郁模型大鼠食欲减退、体重减轻症状的效果仅次于Flu组药物，A组提取物改善CUMS结合孤养抑郁模型大鼠食欲减退、体重减轻症状的效果最差。D组与E组比有显著差异P<0.01。
表2 各组大鼠体重增长量比较(
Figure PCTCN2021109326-appb-000016
n＝10)
Figure PCTCN2021109326-appb-000017
与正常组比较，
Figure PCTCN2021109326-appb-000018
与模型组比较，＊P<0.05，#P<0.01
3.2行为学实验结果
3.2.1糖水消耗百分比结果
各实验组大鼠随着造模过程可见糖水消耗百分比逐渐降低，结果见表3；模型组大鼠糖水消耗百分比较正常组显著下降(P<0.01)，表明CUMS结合孤养造模方法降低大鼠糖水消耗百分比，模拟了抑郁症患者快感缺失症状。
各给药组糖水消耗百分比较模型组均显著升高(P<0.01)，表明各给药组改善CUMS结合孤养抑郁模型大鼠的快感缺失症状。由表可知：总体上各给药组大鼠糖水消耗百分比大小关系依次为Flu组>D组>E组>F组>C组>A组，表明D组提取物改善CUMS结合孤养抑郁模型大鼠快感缺失症状的效果仅次于Flu组药物，A组提取物改善CUMS结合孤养抑郁模型大鼠快感缺失症状的效果最差。D组与E组比有显著差异P<0.01。
表3 各组大鼠糖水消耗百分比结果(
Figure PCTCN2021109326-appb-000019
n＝10)
Figure PCTCN2021109326-appb-000020
与正常组比较，
Figure PCTCN2021109326-appb-000021
与模型组比较，＊P<0.05，#P<0.01
3.2.2敞箱实验结果
(1)水平运动得分
各实验组大鼠敞箱水平运动得分随着造模过程可见逐渐降低，结果见各组大鼠敞箱水平运动得分 结果见表4，模型组大鼠敞箱水平运动得分较正常组显著下降(P<0.01)，表明CUMS结合孤养造模方法可显著降低大鼠的情绪和自主活动能力，模拟了抑郁症患者的自主活动能力低下症状。
各给药组敞箱水平运动得分较模型组均显著升高(P<0.01)，表明各给药组均可有效上调CUMS结合孤养抑郁模型大鼠的情绪和自主活动能力，从而改善CUMS结合孤养抑郁模型大鼠的自主活动能力低下症状。由表可知：总体上各给药组大鼠水平运动得分大小关系依次为Flu组>D组>E组>F组>C组>A组，表明D组提取物改善CUMS结合孤养抑郁模型大鼠的活动能力低下症状的效果仅次于Flu组药物，A组提取物改善CUMS结合孤养抑郁模型大鼠的自主活动能力低下症状的效果最差。D组与E组比有显著差异P<0.01。
表4 各组大鼠敞箱实验水平运动得分结果(
Figure PCTCN2021109326-appb-000022
n＝10)
Figure PCTCN2021109326-appb-000023
与正常组比较，
Figure PCTCN2021109326-appb-000024
与模型组比较，＊P<0.05，#P<0.01
(2)垂直运动得分
各实验组大鼠垂直水平运动得分随着造模过程可见逐渐降低，结果见各组大鼠敞箱垂直运动得分结果见表5，模型组大鼠敞箱垂直运动得分较正常组显著下降(P<0.01)，表明CUMS结合孤养造模方法可显著降低大鼠的空间探索能力，模拟了抑郁症患者对新鲜环境好奇程度降低症状。
各给药组敞箱垂直运动得分较模型组均显著升高(P<0.05)，表明各给药组均可改善CUMS结合孤养抑郁模型大鼠对新鲜环境好奇程度降低症状。由表可知：总体上各给药组大鼠垂直运动得分大小关系依次为Flu组>D组>E组>F组>C组>A组，表明D组提取物改善CUMS结合孤养抑郁模型大鼠对新鲜环境的好奇程度降低症状的效果仅次于Flu组药物，A组提取物改善CUMS结合孤养抑郁模型大鼠对新鲜环境好奇程度降低症状的效果最差。D组与E组比有显著差异P<0.05。
表5 各组大鼠敞箱实验垂直运动得分结果(
Figure PCTCN2021109326-appb-000025
n＝10)
Figure PCTCN2021109326-appb-000026
与正常组比较，
Figure PCTCN2021109326-appb-000027
与模型组比较，＊P<0.05，#P<0.01
3.3.3生化指标检测结果
(1)各组大鼠血浆中CORT含量比较
各组大鼠血浆中CORT含量见表6。模型组大鼠血浆中CORT含量较正常组显著上升(P<0.01)，表明CUMS结合孤养造模方法显著升高大鼠血浆中CORT含量，造成HPA轴功能亢进，从而造成大鼠抑郁。
各给药组大鼠血浆中CORT含量较模型组均显著下降(P<0.01)，表明各给药组均可改善CUMS结合孤养模型抑郁大鼠HPA轴功能亢进，从而发挥抗抑郁作用。由表可知：总体上各给药组大鼠血浆中CORT含量大小关系依次为Flu组>D组>F组>E组>C>组>A组，表明D组提取物抗抑郁作用仅次于Flu组药物，A组提取物抗抑郁作用最差。D组与E组比有显著差异P<0.01。
表6 各组大鼠血浆中CORT含量比较(
Figure PCTCN2021109326-appb-000028
n＝10)
Figure PCTCN2021109326-appb-000029
与正常组比较，
Figure PCTCN2021109326-appb-000030
与模型组比较，＊P<0.05，#P<0.01
(2)各组大鼠皮层中DA、NE、5-HT、BDNF含量比较
各组大鼠皮层中DA、NE、5-HT、BDNF含量见表7。模型组大鼠皮层中DA、NE、5-HT、BDNF含量较正常组显著降低(P<0.01)，表明CUMS结合孤养造模方法显著降低大鼠皮层中单胺类神经递质DA、NE、5-HT含量及脑源性神经营养因子BDNF含量，从而造成大鼠抑郁。
各给药组大鼠皮层中DA、NE、5-HT、BDNF含量较模型组均显著上升(P<0.01)，表明各给药组均可提高CUMS结合孤养抑郁模型大鼠皮层中单胺类神经递质水平，且通过上调皮层中BDNF含量，促进神经元的修复与再生，从而发挥抗抑郁作用。由表可知：总体上各给药组大鼠皮层中DA、NE、5-HT含量大小关系依次为Flu组>D组>F组>E组>C组>A组，各给药组大鼠皮层中BDNF含量大小关系依次为D组>Flu组>F组>E组>C组>A组，表明D组提取物抗抑郁作用最好，A组提取物抗抑郁作用最差。皮层DA含量、NE含量、5-HT中D组与E组比有显著差异P<0.05。BDNF含量D组与E组比有显著差异P<0.01。
表7 各组大鼠皮层中DA、NE、5-HT、BDNF含量比较(
Figure PCTCN2021109326-appb-000031
n＝10)
Figure PCTCN2021109326-appb-000032
Figure PCTCN2021109326-appb-000033
与正常组比较，
Figure PCTCN2021109326-appb-000034
与模型组比较，＊P<0.05，#P<0.01。
(3)各组大鼠海马中DA、NE、5-HT、BDNF含量比较
各组大鼠海马中DA、NE、5-HT、BDNF含量比较见表8。模型组大鼠海马中DA、NE、5-HT、BDNF含量较正常组显著降低(P<0.01)，表明CUMS结合孤养造模方法显著降低大鼠海马中单胺类神经递质DA、NE、5-HT含量及脑源性神经营养因子BDNF含量，从而造成大鼠抑郁。
各给药组大鼠海马中DA、NE、5-HT、BDNF含量较模型组均显著上升(P<0.01)表明各给药组均可提高CUMS结合孤养抑郁模型大鼠海马中单胺类神经递质水平，且通过上调海马中BDNF含量，促进神经元的修复与再生，从而发挥抗抑郁作用。由表可知：总体上各给药组大鼠海马中DA、NE、5-HT含量大小关系依次为Flu组>D组>F组>E组>C组>A组，各给药组大鼠海马中BDNF含量大小关系依次为D组>Flu组>F组>E组>C组>A组，表明D组提取物抗抑郁作用最好，A组提取物抗抑郁作用最差。海马DA含量、NE含量中D组与E组比有显著差异P<0.05；5-HT含量、BDNF含量中D组与E组比有显著差异P<0.01。
表8 各组大鼠海马中DA、NE、5-HT、BDNF含量比较(
Figure PCTCN2021109326-appb-000035
n＝10)
Figure PCTCN2021109326-appb-000036
与正常组比较，
Figure PCTCN2021109326-appb-000037
与模型组比较，＊P<0.05，#P<0.01。
4小结
慢性温和不可预见性应激(CUMS)结合孤养方法建立抑郁模型大鼠，可使大鼠体重增长显著降低、糖水消耗百分比下降、敞箱实验水平运动得分和垂直运动得分减少，成功模拟了临床抑郁症患者体重减轻、快感缺失、自主活动能力及对新鲜环境的好奇程度降低的症状，表明CUMS结合孤养抑郁模型大鼠建立成功。
各组大鼠药效指标结果大小顺序结果见表9。由表可知：总体上各给药组大鼠药效指标结果大小关系依次为Flu组、D组>F组、E组>C组>A组，表明D组提取物抗抑郁作用与Flu组药物相当，A组提取物抗抑郁作用最差。
综上所述，A组、C组、D组、E组、F提取物均可通过抵抗HPA轴功能亢进、提高单胺类神经递质含量及提高神经元可塑性发挥抗抑郁作用。总体上D组提取物抗抑郁作用与Flu组药物相当，A组提取物抗抑郁作用最差。D组与E组比有显著差异。
表9 各组大鼠药效指标结果大小顺序表
药效指标 各组大小顺序
体重增长量 Flu组>D组>F组>E组>C组>A组
糖水消耗百分比 Flu组>D组>E组>F组>C组>A组
水平运动得分 Flu组>D组>E组>F组>C组>A组
垂直运动得分 Flu组>D组>E组>F组>C组>A组
血浆中CORT含量 Flu组>D组>F组>E组>C>组>A组
皮层中DA、NE、5-HT含量 Flu组>D组>F组>E组>C组>A组
皮层中BDNF含量 D组>Flu组>F组>E组>C组>A组
海马中DA、NE、5-HT含量 Flu组>D组>F组>E组>C组>A组
海马中BDNF含量 D组>Flu组>F组>E组>C组>A组
合计 Flu组、D组>E组、F组>C组>A组
试验例二本申请分组提取组合物与合提组合物的对比数据
1实验动物与材料
1.1实验动物
SPF级雄性Sprague Dawley(SD)大鼠90只[购于中国食品药品检定研究院，许可证号：SCXK(京)2017-0005]，体重180～200g，适应性饲养及实验地点均为天津中医药大学实验动物中心。饲养条件：(22±2)℃，湿度(50±10)％，自然光照，自由摄食和饮水。实验动物的饲养和操作规程均遵守天津中医药大学实验动物饲养和使用的相关规定。
1.2药品与试剂
B组(实施例14)、C组、D组、F组同试验例一表1相同。
1.3实验设备与仪器：同试验例一
2实验方法
2.1药物溶液配制
2.1.1氟西汀混悬液的配制
取盐酸氟西汀胶囊(20mg/粒)，除去胶囊壳，加入0.5％CMC-Na溶液研磨配制成1mg/mL的氟西汀混悬液。
2.1.2各组药液的配制
B组、C组、D组、F组提取物每天临床用量分别为3.26g、3.37g、3.23g、2.72g，按15倍临床等效提取物剂量折合成大鼠的剂量分别为4.4010g/kg、4.5495g/kg、4.3605g/kg、3.6720g/kg。
2.2 CUMS结合孤养抑郁模型建立
将90只SD大鼠适应性饲养7天后，在安静环境下对大鼠进行敞箱实验，根据水平运动和垂直运动得分相近的原则筛选大鼠，剔除得分过高或过低的大鼠。将筛选出大鼠随机分成正常组、模型组、Flu组、B组、C组、D组、F组，每组12只。
正常组大鼠每笼4只常规饲养，自由饮水摄食，不接受任何其他额外刺激；其余各组大鼠均每只单笼饲养，每天随机接受以下2种不同应激刺激且相同刺激不可连续出现，从而使大鼠不能预料何种刺激，避免其产生适应性。CUMS刺激因子包括：束缚2h，冰水高台2h，噪音刺激(110dB，30min)，足底电击(电流4mA，50次，每次间隔5s)，45℃烘箱热烘10min，昼夜颠倒24h，潮湿垫料12h，夹尾(距离大鼠尾尖1cm左右，2min)。持续造模，每周测量各组大鼠体重增长量、糖水消耗百分 比及敞箱实验水平运动与垂直运动得分。
2.3药效学测定指标
2.3.1行为学实验
(1)体重变化
实验第0、7、14、21、28、35、42、54天分别对大鼠进行体重测量，记录各组大鼠体重，分析造模过程中各组大鼠每周体重变化及造模前后体重增长量(％)结果。公式如下：
Figure PCTCN2021109326-appb-000038
(2)糖水消耗实验
实验第0、7、14、21、28、35、42、54天对各组大鼠进行糖水消耗实验。实验前3天进行糖水适应，在笼子上放置两瓶1％浓度蔗糖溶液24h，然后放置1％蔗糖溶液一瓶、纯水一瓶24h，上下午调换水瓶位置，第3天禁食禁水24h。正式实验各组大鼠分别放置1％蔗糖溶液一瓶、纯水一瓶，上下午调换水瓶位置，记录各组大鼠24h内蔗糖溶液量(糖水消耗量)及纯水消耗量，计算糖水消耗百分比(％)。公式如下：
Figure PCTCN2021109326-appb-000039
(3)敞箱实验
实验第0、7、14、21、28、35、42、54天对各组大鼠进行敞箱实验。实验所用敞箱大小为100cm×100cm×50cm(长×宽×高)，内侧面黑色，将底面用白线划分成100(10cm×10cm)个大小相同小方格。将每只大鼠依次放在中心固定的方格内，排除人为干扰，记录大鼠3min内活动路线。以实验大鼠穿越底面小方格的数量作为水平运动得分，大鼠每穿越1格(3爪以上跨入)均计为水平运动1分，若实验大鼠沿直线行走，则以每10cm计为1分。以实验大鼠直立次数作为垂直运动得分，大鼠双前肢离开敞箱底面为标志，无论大鼠站立多久，均以大鼠放下双前肢计为垂直运动1分。每只大鼠测试完毕后，彻底清理敞箱，再进行下一只大鼠的测试。记录每只大鼠的水平运动得分及垂直运动得分，分析各组大鼠水平运动得分及垂直运动得分差异。
2.3.2生化指标检测
(1)取材
行为学实验后，大鼠眼眶取血约1.5mL，置于肝素浸润的离心管中，8000r·min -1，4℃，离心10min，取上清液，置于-80℃低温冰箱中保存，用于大鼠血浆中皮质酮(CORT)水平的测定。
眼眶取血后，脱颈处死，在冰面上迅速剥离出皮层和海马，称重，置冻存管中在液氮中迅速冷冻，然后转移至-80℃低温冰箱中保存，用于大鼠皮层与海马中神经生长营养因子(BDNF)、5-羟色胺(5-HT)、多巴胺(DA)和去甲肾上腺素(NE)含量的测定。
(2)CORT、BDNF、5-HT、DA、NE测定及计算
采用酶联免疫吸附测定(Enzyme linked immunosorbent assay，ELISA)对各组大鼠血浆中CORT含量及皮层与海马中BDNF、5-HT、DA和NE含量进行测定，在450nm波长下依序测量各孔的吸光度(OD值)。以标准物的含量为横坐标，OD值为纵坐标，绘制标准曲线并得回归方程，将各组大鼠 血浆中CORT及皮层与海马中BDNF、5-HT、DA和NE的OD值代入回归方程，计算各组大鼠血浆中CORT及皮层与海马中BDNF、5-HT、DA和NE的含量。
2.4.实验数据处理及统计学分析
实验数据以
Figure PCTCN2021109326-appb-000040
表示，采用SPSS 22.0统计学软件分析，大鼠血浆中CORT含量及皮层与海马中BDNF、5-HT、DA、NE含量采用单因素方差分析(One-way ANOVA)进行组间差异分析。
3实验结果与分析
3.1行为学实验结果
3.1.1体重变化结果
各实验组大鼠随着造模过程可见不同程度的体重增加，结果见各组大鼠体重增长量结果见表10，模型组大鼠体重增长量较正常组显著下降(P<0.01)，表明CUMS结合孤养造模方法影响大鼠的食欲导致体重增长缓慢，模拟了抑郁症患者食欲减退、体重减轻症状。
表10 各组大鼠体重增长量比较(
Figure PCTCN2021109326-appb-000041
n＝10)
Figure PCTCN2021109326-appb-000042
与正常组比较，
Figure PCTCN2021109326-appb-000043
与模型组比较，＊P<0.05，#P<0.01
各给药组大鼠体重增长量较模型组显著升高(P<0.01)，Flu组大鼠体重增长量高于D组，但Flu组与D组大鼠体重增长量比较无显著性差异(P>0.05)，其它各组大鼠体重增长量均有显著性差异(P<0.05)，表明各给药组均可抵抗CUMS结合孤养抑郁模型大鼠体重降低，从而改善CUMS结合孤养抑郁模型大鼠食欲减退、体重减轻症状。由表10可知，各给药组大鼠体重增长量大小关系依次为Flu组，D组>B组>F组>C组，表明D组提取物改善CUMS结合孤养抑郁模型大鼠食欲减退、体重减轻症状的效果与Flu组药物相当，C组提取物改善CUMS结合孤养抑郁模型大鼠食欲减退、体重减轻症状的效果最差。D组与B组比有显著差异P<0.01。
3.1.2糖水消耗百分比结果
各实验组大鼠随着造模过程可见糖水消耗百分比逐渐降低，结果见各组大鼠糖水消耗比结果见表11，模型组大鼠糖水消耗百分比较正常组显著下降(P<0.01)，表明CUMS结合孤养造模方法降低大鼠糖水消耗百分比，模拟了抑郁症患者快感缺失症状。
表11 各组大鼠糖水消耗百分比结果(
Figure PCTCN2021109326-appb-000044
n＝10)
Figure PCTCN2021109326-appb-000045
Figure PCTCN2021109326-appb-000046
与正常组比较，
Figure PCTCN2021109326-appb-000047
与模型组比较，＊P<0.05，#P<0.01
与模型组比较，各给药组糖水消耗百分比均显著升高(P<0.01)，Flu组大鼠糖水消耗百分比高于D组，但比较Flu组与D组大鼠糖水消耗百分比无显著性差异(P>0.05)，B组大鼠糖水消耗百分比高于F组，但比较B组与F组大鼠糖水消耗百分比无显著性差异(P>0.05)，且其它各组大鼠糖水消耗百分比均有显著性差异((P<0.05))，表明各给药组均可改善CUMS结合孤养抑郁模型大鼠的快感缺失症状。由表11可知：各给药组大鼠糖水消耗百分比大小关系依次为Flu组，D组>B组，F组>C组，表明D组提取物改善CUMS结合孤养抑郁模型大鼠快感缺失症状的效果与Flu组药物相当，C组提取物改善CUMS结合孤养抑郁模型大鼠快感缺失症状的效果最差。D组与B组比有显著差异P<0.05。
3.1.3敞箱实验结果
(1)水平运动得分
各实验组大鼠敞箱水平运动得分随着造模过程可见逐渐降低；各组大鼠敞箱水平运动得分结果见表12，模型组大鼠敞箱水平运动得分较正常组显著下降(P<0.01)，表明CUMS结合孤养造模方法可显著降低大鼠的情绪和自主活动能力，模拟了抑郁症患者的自主活动能力低下症状。
表12 各组大鼠敞箱实验水平运动得分结果(
Figure PCTCN2021109326-appb-000048
n＝10)
Figure PCTCN2021109326-appb-000049
与正常组比较，
Figure PCTCN2021109326-appb-000050
与模型组比较， ＊P<0.05， #P<0.01
给药组敞箱水平运动得分较模型组均显著升高(P<0.01)，Flu组大鼠敞箱水平运动得分高于D组，但Flu组与D组大鼠敞箱水平运动得分比较无显著性差异(P>0.05)，且其它各组大鼠敞箱运动得分均有显著性差异(P<0.05)，表明各给药组均可有效上调CUMS结合孤养抑郁模型大鼠的情绪和自主活动能力，从而改善CUMS结合孤养抑郁模型大鼠的自主活动能力低下症状。由表12可知：各给药组大鼠水平运动得分大小关系依次为Flu组，D组>B组>F组>C组，表明D组提取物改善CUMS结合孤养抑郁模型大鼠的活动能力低下症状的效果与Flu组药物相当，C组提取物改善CUMS结合孤养抑郁模型大鼠的自主活动能力低下症状的效果最差。D组与B组比有显著差异P<0.01。
(2)垂直运动得分
各实验组大鼠垂直水平运动得分随着造模过程可见逐渐降低；各组大鼠敞箱垂直运动得分结果见表14，模型组大鼠敞箱垂直运动得分较正常组显著下降(P<0.01)，表明CUMS结合孤养造模方法可显著降低大鼠的空间探索能力，模拟了抑郁症患者对新鲜环境好奇程度降低症状。
表14 各组大鼠敞箱实验垂直运动得分结果(
Figure PCTCN2021109326-appb-000051
n＝10)
Figure PCTCN2021109326-appb-000052
Figure PCTCN2021109326-appb-000053
与正常组比较，
Figure PCTCN2021109326-appb-000054
与模型组比较， ＊P<0.05， #P<0.01
各给药组敞箱垂直运动得分较模型组均显著升高(P<0.05)，且其它各组大鼠敞箱垂直运动得分均有显著性差异(P<0.05)，表明各给药组均可改善CUMS结合孤养抑郁模型大鼠对新鲜环境好奇程度降低症状。由表14可知：各给药组大鼠垂直运动得分大小关系依次为Flu组>D组>B组>F组>C组，表明D组提取物改善CUMS结合孤养抑郁模型大鼠对新鲜环境的好奇程度降低症状的效果仅次于Flu组药物，C组提取物改善CUMS结合孤养抑郁模型大鼠对新鲜环境好奇程度降低症状的效果最差。D组与B组比有显著差异P<0.05。
3.2生化指标检测结果
3.2.1各组大鼠血浆中CORT含量比较
皮质酮(CORT)是一种糖皮质激素，高浓度的皮质酮与其受体结合产生神经毒性，造成海马区神经元损伤，导致下丘脑-垂体-肾上腺(HPA)轴功能亢进，从而引起抑郁。
各组大鼠血浆中CORT含量见表15。模型组大鼠血浆中CORT含量较正常组显著上升(P<0.01)，表明CUMS结合孤养造模方法显著升高大鼠血浆中CORT含量，造成HPA轴功能亢进，从而造成大鼠抑郁。
表15 各组大鼠血浆中CORT含量比较(
Figure PCTCN2021109326-appb-000055
n＝10)
Figure PCTCN2021109326-appb-000056
与正常组比较，
Figure PCTCN2021109326-appb-000057
与模型组比较， ＊P<0.05， #P<0.01
各给药组大鼠血浆中CORT含量较模型组均显著下降(P<0.01)，且其它各组大鼠血浆中CORT含量均有显著性差异(P<0.05)，表明各给药组均可改善CUMS结合孤养模型抑郁大鼠HPA轴功能亢进，从而发挥抗抑郁作用。由表15可知：各给药组大鼠血浆中CORT含量大小关系依次为Flu组<D组<B组<F组<C组，表明D组提取物抗抑郁作用仅次于Flu组药物，C组提取物抗抑郁作用最差。D组与B组比有显著差异P<0.05。
3.2.2各组大鼠皮层及海马中DA、NE、5-HT、BDNF含量比较
多巴胺(DA)、去甲肾上腺素(NE)和5-羟色胺(5-HT)是大脑中枢神经系统单胺类神经递质，脑源性神经营养因子(BDNF)是一种对神经元具有营养支持作用的蛋白质，其水平下降可导致抑郁症发生。
各组大鼠皮层中DA、NE、5-HT、BDNF含量见表16。各组大鼠海马中DA、NE、5-HT、BDNF含量比较见表17。模型组大鼠皮层及海马中DA、NE、5-HT、BDNF含量较正常组显著降低(P<0.01)，表明CUMS结合孤养造模方法显著降低大鼠皮层及海马中单胺类神经递质DA、NE、5-HT含量及脑源性神经营养因子BDNF含量，从而造成大鼠抑郁。
表16 各组大鼠皮层中DA、NE、5-HT、BDNF含量比较(
Figure PCTCN2021109326-appb-000058
n＝10)
Figure PCTCN2021109326-appb-000059
与正常组比较，
Figure PCTCN2021109326-appb-000060
与模型组比较， ＊P<0.05， #P<0.01
表17 各组大鼠海马中DA、NE、5-HT、BDNF含量比较(
Figure PCTCN2021109326-appb-000061
n＝10)
Figure PCTCN2021109326-appb-000062
与正常组比较，
Figure PCTCN2021109326-appb-000063
与模型组比较， ＊P<0.05， #P<0.01
各给药组大鼠皮层及海马中DA含量较模型组均显著上升(P<0.01)，D组大鼠皮层DA含量与B组无显著性差异(P>0.05)，且其它各组大鼠皮层及海马中DA含量均有显著性差异(P<0.05)，表明各给药组均可提高CUMS结合孤养抑郁模型大鼠皮层及海马中DA含量，从而发挥抗抑郁作用。由表15-16可知：各给药组大鼠皮层中DA含量大小关系依次为Flu组>D组，B组>F组>C组，B相对D有显著性差异P>0.05。
各给药组大鼠海马中DA含量大小关系依次为Flu组>D组>B组>F组>C组，D相对B有显著性差异P<0.01。以上表明D组提取物抗抑郁作用仅次于Flu组药物，C组提取物抗抑郁作用最差。
各给药组大鼠皮层及海马中NE含量较模型组均显著上升(P<0.01)，Flu组大鼠皮层及海马中NE含量与D组无显著性差异(P>0.05)，且其它各组大鼠皮层及海马中NE含量均有显著性差异(P<0.05)，表明各给药组均可提高CUMS结合孤养抑郁模型大鼠皮层及海马中NE含量，从而发挥抗抑郁作用。由表15-16可知：各给药组大鼠皮层及海马中NE含量大小关系依次为Flu组，D组>B组>F组>C组，D相对B有显著性差异。表明D组提取物抗抑郁作用与Flu组药物相当，C组提取物抗抑郁作用最差。
各给药组大鼠皮层及海马中5-HT含量较模型组均显著上升(P<0.05)，D组大鼠皮层5-HT含量与B组无显著性差异(P>0.05)，B组大鼠海马5-HT含量与F组无显著性差异(P>0.05)，且其它各 组大鼠皮层及海马中5-HT含量均有显著性差异(P<0.05)，表明各给药组均可提高CUMS结合孤养抑郁模型大鼠皮层及海马中5-HT含量，从而发挥抗抑郁作用。由表15-16可知：各给药组大鼠皮层中5-HT含量大小关系依次为Flu组>D组，B组>F组>C组，B相对D有显著性差异P<0.05；各给药组大鼠海马中5-HT含量大小关系依次为Flu组>D组>B组，F组>C组，D相对B有显著性差异P<0.05。表明D组提取物抗抑郁作用仅次于Flu组药物，C组提取物抗抑郁作用最差。
各给药组大鼠皮层及海马中BDNF含量较模型组均显著上升(P<0.01)，Flu组大鼠皮层中BDNF含量与D组无显著性差异(P>0.05)，且其它各组大鼠皮层及海马中BDNF含量均有显著性差异(P<0.05)，表明各给药组均可提高CUMS结合孤养抑郁模型大鼠皮层及海马中BDNF含量，促进神经元的修复与再生，从而发挥抗抑郁作用。由表15-16可知：各给药组大鼠皮层中BDNF含量大小关系依次为Flu组，D组>B组>F组>C组，各给药组大鼠皮层及海马中NE含量大小关系依次为Flu组>D组>B组>F组>C组，D相对B有显著性差异P<0.01，表明D组提取物抗抑郁作用仅次于Flu组药物，C组提取物抗抑郁作用最差。
4小结
4.1抑郁模型大鼠建立
慢性温和不可预见性刺激(CUMS)结合孤养方法建立抑郁模型大鼠后，模型组大鼠行为学指标(体重变化、糖水消耗百分比、敞箱实验运动得分)较正常组显著降低；生化指标DA、NE、5-HT、BDNF较正常组显著降低，而CORT较正常组显著升高，表明抑郁模型大鼠造模成功。
4.2行为学和生化指标测定结果
各给药组大鼠行为学指标(体重增长量、糖水消耗百分比、敞箱实验运动得分)和生化指标(DA、NE、5-HT、BDNF)较模型组均显著升高，而血浆CORT较模型组均显著降低，表明各给药组均有抗抑郁作用。
各给药组大鼠行为学及生化指标大小顺序结果见表18。由表可知：D组提取物抗抑郁作用最好，C组提取物抗抑郁作用最差。
表18 各组大鼠行为学及生化指标结果大小顺序表
Figure PCTCN2021109326-appb-000064
综上所述，本研究抑郁模型大鼠造模成功后，行为学指标和生化指标测定结果中，虽某些组间无显著性差异，但总体上各给药组大鼠行为学指标和皮层及海马中生化指标含量大小顺序为Flu组>D组>B组>F组>C组，血浆中CORT含量大小顺序为Flu组<D组<B组<F组<C组。
D组、B组、F组、C组提取物均可通过抵抗HPA轴功能亢进、提高单胺类神经递质含量及提高神经元可塑性而发挥抗抑郁作用。其中D组和B组提取物行为学指标和生化指标均高于F组；D组和B组总趋势为D组>B组，但皮层中5-HT和DA含量无显著性差异，其它指标D组和B组有显著差异；B组和F组总趋势为B组>F组，但糖水消耗百分比、皮层及海马中NE含量、海马中5-HT含量无显著性差异；C组提取物抗抑郁作用最差。
实施例32：5味药材分组方式考察
实验目的：5味药材水合提得到的提取物，在质量控制时发现柴胡成分对当归中阿魏酸定量有干扰，为解决这个问题，特别设计分组提取以避免干扰。
实验方法：5味药材分组提取设计主要依据3个原则：1、柴胡与当归分开，避免柴胡成分对当归中阿魏酸定量的干扰；2、每组提取物中至少含有1个稳定的指标性成分以表征提取物到制剂过程的工艺稳定性；3、在提取过程收集芳香水，在浓缩收膏终点将芳香水加入提取物中，使整体提取物与传统应用水煎剂成分接近。
实验结果：
1.收油角度的分组方式：
减方后，处方里还有柴胡、当归、麸炒白术、甘草、白芍共5味，其中有挥发性成分的是柴胡、当归和麸炒白术。考察单提出油情况：柴胡出油量极少，无法收集，约0.2ml/kg；麸炒白术有少量的油，0.95ml/kg；当归出油较明显，4.3ml/kg。
从挥发油收集的角度，当归和麸炒白术可以一起收集挥发油，柴胡因为油量极少，进行合并水提。
2.分析Marker角度的分组方式：
目的：筛选当归和麸炒白术组中合适的分析标记物来表征提取物到制剂过程的稳定性。
当归：筛选出的藁本内酯和阿魏酸都不适合做分析标记物。藁本内酯是挥发性成分，受挥发油收集状态影响，提取批次间波动较大，且挥发性成分稳定性风险高。阿魏酸含量水平低(0.1％左右)且性质不稳定(对高温和光敏感)，在提取物中可定量，但在制剂中受柴胡成分的干扰，不能准确定量。
麸炒白术：筛选出的苍术酮和白术内酯Ⅲ成分都不适合做分析标记物。苍术酮为挥发性成分，受挥发油收集状态影响，提取批次间波动较大，且挥发性成分稳定性风险高。白术内酯Ⅲ含量水平低(0.01％左右)，制剂中的可检测性差，从提取物到制剂的转移率存在风险。
考虑在当归和麸炒白术组加入甘草，将甘草酸成分作为该组提取物的分析标记物来表征提取物到制剂的过程稳定性。因为甘草酸性质较为稳定，不受长时间煎煮的影响，见图3，且甘草酸处方比例低，有利于两组提取物之间的批次对应。
实验结论：
实验考察后确定的提取分组为：柴胡、白芍合并提取，得到逍遥柴白提取物；当归、麸炒白术、炙甘草合并提取得到逍遥当白甘提取物。如图2所示，包含当归的逍遥当白甘提取物，阿魏酸成分的含量分析方法专属性合格，提取物可实现全面的质量控制。如图3所示，甘草酸成分稳定可以作为该组提取物的分析标记物来表征提取物到制剂的过程稳定性。

Claims (10)

  1. 一种具有缓解精神及抗抑郁功效的中药组合物，所述中药组合物由柴胡，白芍，麸炒白术，当归，炙甘草以及任选的加入或不加入茯苓组成的重量份中药原料药配方制备而成，其中不加入茯苓的配方为配方1：
    柴胡10～30
    白芍10～30
    麸炒白术15～30
    当归10～30
    炙甘草15～24
    其中加入茯苓的配方为配方2：
    柴胡10～30
    白芍10～30
    麸炒白术15～30
    当归10～30
    茯苓10～30
    炙甘草15～24。
  2. 根据权利要求1所述的中药组合物，其中，所述配方1为：
    柴胡15～25
    白芍15～25
    麸炒白术15～25
    当归15～25
    炙甘草15～20
    其中所述配方2为：
    柴胡15～25
    白芍15～25
    麸炒白术15～25
    当归15～25
    茯苓15～25
    炙甘草15～20。
  3. 根据权利要求1所述的中药组合物，其中，所述配方1为：
    柴胡20
    白芍20
    麸炒白术20
    当归20
    炙甘草16
    其中所述配方2为：
    柴胡20
    白芍20
    麸炒白术20
    当归20
    茯苓20
    炙甘草16。
  4. 根据权利要求1-3中任一项所述的中药组合物，其中，所述中药组合物通过包括如下步骤的方法制备：按组方配比称取药材，加入4～7倍重量的水，提取1-3次，每次1～3h，提取后滤过，合并两次提取的滤液，静置4h以上，取上清液，浓缩得到提取物；以提取物为药物活性成分，必要时加入药用辅料，制备成药物制剂中药组合物；
    优选，所述中药组合物通过包括如下步骤的方法制备：按组方配比称取药材，加入6-7倍重量的水，提取2-3h，过滤，药渣加入5-6倍重量的水，提取1h，过滤，合并两次提取的滤液，静置4h以上，取上清液，浓缩，浓缩终点控制在糖度65％～70％，以提取物为药物活性成分，必要时加入药用辅料，制备成药物制剂中药组合物。
  5. 根据权利要求1-3中任一项所述的中药组合物，其中，所述配方1的中药组合物中包括折干重量逍遥柴白提取物9份，逍遥当白甘提取物32份；其中所述逍遥柴白提取物中柴胡皂苷B2折干含量0.63～10.0mg/g、芍药苷折干含量9.4～115.9mg/g；所述逍遥当白甘提取物中甘草酸折干含量1.4～26.1mg/g、阿魏酸折干含量0.13～0.91mg/g、白术内酯III折干含量0.0038～0.30mg/g；所述逍遥柴白提取物是由柴胡、白芍提取得到的；所述逍遥当白甘提取物是由麸炒白术、当归、炙甘草提取得到的。
  6. 根据权利要求5所述的中药组合物，其中，所述逍遥柴白提取物中柴胡皂苷折干含量B20.94～6.8mg/g、芍药苷折干含量17.6～110.9mg/g；所逍遥当白甘提取物中甘草酸折干含量6.9～26.1mg/g、阿魏酸折干含量0.16～0.79mg/g、白术内酯III折干含量0.0047～0.25mg/g。
  7. 根据权利要求5所述的中药组合物，其中，所述中药组合物通过包括如下步骤的方法制备：
    (1)逍遥柴白提取物的提取：在柴胡、白芍药材中加入4～7倍重量的水，提取1～3次，每次1～3h，提取后滤过，合并两次提取的滤液，静置4h以上，取上清液，浓缩即得；
    (2)逍遥当白甘提取物的提取：在当归、麸炒白术、炙甘草药材中加入4～7倍重量的水，提取1～3次，每次1～3h，提取结束收集挥发油和芳香水，提取后提取液过滤，合并两次提取的提取液静置4h以上，取上清液，浓缩，浓缩，加入所收集的挥发油和芳香水，再次浓缩即得；和
    (3)取逍遥柴白提取物和逍遥当白甘提取物按照配比混合均匀，即得。
  8. 根据权利要求7所述的中药组合物，其中，所述中药组合物通过包括如下步骤的方法制备：
    (1)逍遥柴白提取物的提取：在柴胡、白芍药材中加入6倍重量的水，提取2h，过滤，药渣加入5倍重量的水，提取1h，过滤，合并两次提取的滤液，静置4h以上，取上清液，浓缩，浓缩终点密度控制在1.26～1.28，即得；
    (2)逍遥当白甘提取物的提取：在当归、麸炒白术、炙甘草药材中加入6倍重量的水，提取2h，过滤，药渣加入5倍重量的水，提取1h，提取结束收集挥发油和芳香水，提取液过滤，合并两次提取的提取液静置4h以上，取上清液，浓缩，浓缩至终点，加入所收集的挥发油和芳香水，再次浓缩至终点，浓缩终点密度控制在1.29～1.31；和
    (3)取逍遥柴白提取物和逍遥当白甘提取物按照配比混合均匀，即得。
  9. 根据权利要求5所述的中药组合物，其中，所述中药组合物通过包括如下步骤的方法制备：
    (1)逍遥柴白提取物的提取：在柴胡、白芍药材中加入4～7倍重量的水，提取1～3次，每次1～3h，提取后滤过，合并两次提取的滤液，静置4h以上，取上清液，浓缩即得；
    (2)逍遥当白甘提取物的提取：在当归、麸炒白术、炙甘草药材中加入4～7倍重量的水，提取1～3次，每次1～3h，提取结束收集挥发油和芳香水，提取后提取液过滤，合并两次提取的提取液静置4h以上，取上清液，浓缩，浓缩，加入所收集的挥发油和芳香水，再次浓缩即得；和
    (3)取逍遥柴白提取物和逍遥当白甘提取物按照配比混合均匀，以逍遥柴白提取物和逍遥当白甘提取物为药物活性成分，必要时加入药用辅料，制备成药物制剂中药组合物；
    优选包括如下步骤：
    逍遥柴白提取物的提取：在柴胡、白芍药材中加入6倍重量的水，提取2h，过滤，药渣加入5倍重量的水，提取1h，过滤，合并两次提取的滤液，静置4h以上，取上清液，浓缩，浓缩终点密度控制在1.26～1.28，即得；
    逍遥当白甘提取物的提取：在当归、麸炒白术、炙甘草药材中加入6倍重量的水，提取2h，过滤，药渣加入5倍重量的水，提取1h，提取结束收集挥发油和芳香水，提取液过滤，合并两次提取的提取液静置4h以上，取上清液，浓缩，浓缩至终点，加入所收集的挥发油和芳香水，再次浓缩至终点，浓缩终点密度控制在1.29～1.31；和
    取逍遥柴白提取物和逍遥当白甘提取物按照配比混合均匀，以逍遥柴白提取物和逍遥当白甘提取物为药物活性成分，必要时加入药用辅料，制备成药物制剂中药组合物。
  10. 根据权利要求4或9所述的中药组合物，其中，所述药物制剂中药组合物是片剂，胶囊，颗粒，口服液，滴丸或丸剂。
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