WO2021063391A1 - 一种山羊角角蛋白、其制备方法、其药物组合物及用途 - Google Patents

一种山羊角角蛋白、其制备方法、其药物组合物及用途 Download PDF

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WO2021063391A1
WO2021063391A1 PCT/CN2020/119336 CN2020119336W WO2021063391A1 WO 2021063391 A1 WO2021063391 A1 WO 2021063391A1 CN 2020119336 W CN2020119336 W CN 2020119336W WO 2021063391 A1 WO2021063391 A1 WO 2021063391A1
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goat
keratin
content
extract
preparation
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PCT/CN2020/119336
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English (en)
French (fr)
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庾石山
王晓良
屈晶
符江
史国茹
王玲
冯楠
徐少峰
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中国医学科学院药物研究所
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4741—Keratin; Cytokeratin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/32—Bones; Osteocytes; Osteoblasts; Tendons; Tenocytes; Teeth; Odontoblasts; Cartilage; Chondrocytes; Synovial membrane
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A61P11/10—Expectorants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A61P11/14—Antitussive agents
    • 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
    • A61P25/08—Antiepileptics; Anticonvulsants
    • 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
    • A61P25/20—Hypnotics; Sedatives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/12—Antihypertensives
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/30—Extraction; Separation; Purification by precipitation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/34—Extraction; Separation; Purification by filtration, ultrafiltration or reverse osmosis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/36—Extraction; Separation; Purification by a combination of two or more processes of different types
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the invention belongs to the technical field of medicine, and relates to a goat keratin, a preparation method thereof, a pharmaceutical composition containing the keratin, and the preparation of the keratin and the pharmaceutical composition for antipyretic, analgesic, anticonvulsant, and analgesic Application in cough, expectorant, lowering blood pressure, anti-inflammatory and antiviral drugs.
  • Goat horns are the horns of Bovidae (Bovidae) animals of the genus Capra (Naemorhedus goral Hardwicke) and Ibex (Capra ibex Linnaeus). They are cold, salty in nature, home to heart, and liver meridian.
  • goat horns are all replaced by goat horns, indicating that the use of goat horns instead of antelope horns is increasingly allowed and recognized, but the amount of goat horns Compared with antelope horns, there are obvious differences. Therefore, the research on goat horns, especially the research on keratin components that play a major role, is more worthy of in-depth exploration and development to ensure the safety and convenience of goat horns in clinical applications. Sex, resource sustainability. On this basis, we extracted and purified goat keratin to prepare goat keratin, laying the foundation for subsequent development and utilization.
  • the technical problem solved by the present invention is to provide a kind of goat keratin, its preparation method, pharmaceutical composition and application.
  • the present invention adopts the following technical solutions:
  • the first aspect of the technical scheme of the present invention is to provide a method for preparing goat keratin keratin, which is characterized in that the preparation method is as follows:
  • the preferred raw medicinal material of goat horn is goat horn after removing the plug and washing.
  • the most preferred goat horn is dried and appropriately pulverized to increase the contact area with the solvent and improve the extraction efficiency.
  • Goat horn fine powder needs to be cold leached first.
  • the solvent for cold leaching extraction is water.
  • the dregs after cold leaching extraction are reductively extracted.
  • the solvent for the reduction and extraction is an aqueous solution of inorganic salts.
  • the goat keratin extract obtained in step (1) needs a step of removing impurities.
  • the selected impurity removal step specifically includes: adding inorganic salt to the goat keratin extract solution for impurity removal to obtain the goat keratin extract solution after impurity removal.
  • the selected inorganic salt is potassium chloride.
  • the de-impurity goat keratin extract prepared in step (2) needs the steps of concentration and purification.
  • the steps of concentration and purification include the method of inorganic salt salting out, the method of ultrafiltration of ceramic membrane ultrafiltration system and the method of dialysis.
  • the selected inorganic salt is ammonium sulfate, sodium sulfate, magnesium chloride, magnesium sulfate, aluminum sulfate, ammonium nitrate, ammonium chloride, sodium chloride or their aqueous solutions, preferably an aqueous solution of ammonium sulfate.
  • the second aspect of the technical solution of the present invention is to provide goat keratin keratin prepared by the preparation method described in the first aspect. Further, the type and content of the obtained goat keratin were determined.
  • the content of protein Q0VD04 was 0.00-20.00%; the content of Q14818 was 0.00-20.00%; the content of A5PJJ1 was 0.00-20.00%; the content of F1MXG6 was 0.00 ⁇ 20.00%; F1MC11 content is 0.00 ⁇ 20.00%; G3X7V4 content is 0.00 ⁇ 20.00%; M0QVY0 content is 0.00 ⁇ 20.00%; Q08D91 content is 0.00 ⁇ 20.00%; A0A140T867 content is 0.00 ⁇ 30.00%; The content of F1N362 is 0.00-20.00%; the content of E1B898 is 0.00-50.00%; the content of F1M198 is 0.00-30.00%; the content of Q5XQN5 is 0.00-25.00%.
  • the type and content of the obtained goat keratin are determined, wherein the content of protein Q0VD04 is 0.00-30.00%; the content of Q14818 is 0.00-20.00%; the content of A5PJJ1 is 0.00-40.00%; the content of A3KN26 is 0.00 ⁇ 50.00%; F1MC11 content is 0.00 ⁇ 20.00%; G3X7V4 content is 0.00 ⁇ 20.00%; M0QVY0 content is 0.00 ⁇ 20.00%; Q08D91 content is 0.00 ⁇ 50.00%; F1N362 content is 0.00 ⁇ 20.00%; The content of E1B898 is 0.00-50.00%; the content of F1M198 is 0.00-30.00%; the content of Q5XQN5 is 0.00-20.00%.
  • the third aspect of the technical solution of the present invention is to provide a pharmaceutical composition, which comprises the goat keratin described in the second aspect as an active ingredient and a pharmaceutically acceptable carrier or excipient.
  • the pharmaceutical composition of the present invention can be prepared according to methods known in the art.
  • the goat keratin of the present invention can be combined with one or more solid or liquid pharmaceutical excipients and/or adjuvants to prepare an appropriate administration that can be used as a human or veterinary medicine.
  • Form or dosage form can be prepared according to methods known in the art.
  • the goat keratin or the pharmaceutical composition containing it of the present invention can be administered in a unit dosage form.
  • the route of administration can be enteral or parenteral, such as oral, intramuscular, subcutaneous, nasal, oral mucosa, eyes, lungs, skin, The vagina, peritoneum, rectum, etc. are preferably administered orally.
  • the dosage form for administration may be a liquid dosage form, a solid dosage form or a semi-solid dosage form.
  • Liquid dosage forms can be solutions (including true solutions and colloidal solutions), emulsions (including oil-in-water, water-in-oil and double emulsions), suspensions, injections (including water injections, powder injections and infusions), eye drops Lotion, nasal drops, lotion and liniment, etc.
  • the solid dosage form can be tablets (including ordinary tablets, enteric-coated tablets, buccal tablets, dispersible tablets, chewable tablets, effervescent tablets, orally disintegrating tablets), capsules (including hard capsules, soft capsules, and enteric-coated capsules), granules Preparations, powders, pellets, dripping pills, suppositories, films, patches, air (powder) sprays, sprays, etc.; semi-solid dosage forms can be ointments, gels, pastes, etc.
  • the goat keratin keratin of the present invention can be prepared into ordinary preparations, slow-release preparations, controlled-release preparations, targeted preparations and various particle delivery systems.
  • diluents can be starch, dextrin, sucrose, glucose, lactose, mannitol, sorbitol, xylitol, microcrystalline cellulose, calcium sulfate, calcium hydrogen phosphate, calcium carbonate, etc.
  • the humectant can be water, ethanol, iso Propanol, etc.
  • the binder can be starch syrup, dextrin, syrup, honey, glucose solution, microcrystalline cellulose, acacia syrup, gelatin syrup, sodium carboxymethyl cellulose, methyl cellulose, hypromellose Base cellulose, ethyl cellulose, acrylic resin, carbomer, polyvinylpyrrolidone, polyethylene dipropanol, etc.
  • disintegrant can be dry starch, micro
  • the tablets can also be further made into coated tablets, such as sugar-coated tablets, film-coated tablets, enteric-coated tablets, or double-layer tablets and multi-layer tablets.
  • carriers In order to make the administration unit into a pill, various carriers known in the art can be widely used.
  • carriers are, for example, diluents and absorbents, such as glucose, lactose, starch, cocoa butter, hydrogenated vegetable oil, polyvinylpyrrolidone, polyethylene glycol laurate, kaolin, talc, etc.; binders, such as Gum arabic, xanthan gum, gelatin, ethanol, honey, liquid sugar, rice paste or batter, etc.; disintegrants, such as agar powder, dried starch, alginate, sodium lauryl sulfonate, methyl cellulose, Ethyl cellulose and so on.
  • diluents and absorbents such as glucose, lactose, starch, cocoa butter, hydrogenated vegetable oil, polyvinylpyrrolidone, polyethylene glycol laurate, kaolin, talc, etc.
  • binders such as Gum arabic, xanthan gum, ge
  • various carriers known in the art can be widely used.
  • carriers are, for example, polyethylene glycol, lecithin, cocoa butter, higher alcohols, higher alcohol esters, gelatin, semi-synthetic glycerides and the like.
  • goat keratin or a pharmaceutical composition containing goat keratin is mixed with the above-mentioned various carriers, and the resulting mixture is placed in a hard gelatin capsule or a soft capsule.
  • the active ingredient of the goat keratin of the present invention can also be made into microcapsules, suspended in an aqueous medium to form a suspension, or filled into hard capsules for application.
  • the goat keratin of the present invention is prepared into a solution, a suspension solution, an emulsion, a lyophilized powder injection, and this preparation may be aqueous or non-aqueous, and may contain one and/or more pharmacodynamics.
  • Acceptable carriers, diluents, binders, lubricants, preservatives, surfactants or dispersants can be selected from water, ethanol, polyethylene glycol, 1,3-propanediol, ethoxylated isostearyl alcohol, polyoxylated isostearyl alcohol, polyoxyethylene sorbitol fatty acid esters and the like.
  • an appropriate amount of sodium chloride, glucose or glycerin can be added to the injection preparation, and in addition, conventional solubilizers, buffers, pH adjusters, etc. can also be added. These auxiliary materials are commonly used in this field.
  • coloring agents can also be added to the pharmaceutical preparations.
  • the goat keratin or the pharmaceutical composition of the present invention can be administered by any known administration method.
  • the dosage of the goat keratin pharmaceutical composition of the present invention depends on many factors, such as the nature and severity of the disease to be prevented or treated, the gender, age, weight, personality and individual response of the patient or animal, the route of administration, The number of medicines and the purpose of treatment, so the therapeutic dose of the present invention can have a wide range of changes. Generally speaking, the dosage of the pharmaceutical ingredients of the present invention is well known to those skilled in the art. According to the actual amount of the drug contained in the final preparation of the goat keratin keratin composition of the present invention, appropriate adjustments can be made to achieve the requirement of the therapeutically effective dose and accomplish the preventive or therapeutic purpose of the present invention.
  • the daily suitable dosage range of goat keratin of the present invention is 0.01-500 mg/Kg body weight, preferably 0.5-100 mg/Kg body weight, more preferably 1-50 mg/Kg body weight, most Preferably it is 2-30 mg/Kg body weight.
  • the above-mentioned dosage can be administered in a single dosage form or divided into several, for example, two, three or four dosage forms, depending on the clinical experience of the administering doctor and the dosage regimen including the use of other treatment means.
  • the total dose required for each treatment can be divided into multiple or single doses.
  • the goat keratin or the pharmaceutical composition of the present invention can be taken alone, or combined with other therapeutic drugs or symptomatic drugs, and the dosage is adjusted.
  • the fourth aspect of the technical scheme of the present invention is to provide an application of goat keratin in the preparation of antipyretic, analgesic, anticonvulsant, antitussive, expectorant, blood pressure, anti-inflammatory, and antiviral drugs.
  • the fourth aspect of the technical solution of the present invention also provides that the goat keratin described in the second aspect and the pharmaceutical composition described in the third aspect are used in the preparation of antipyretic, analgesic, anticonvulsant, antitussive, expectorant, blood pressure, Application of anti-inflammatory and antiviral drugs.
  • the medicinal materials are easy to obtain, the extraction process is simple, and it is suitable for large-scale industrial production.
  • Figure 1 The preparation flow chart of goat keratin by salting out method.
  • Figure 2 Flow chart of preparation of goat keratin by ultrafiltration method.
  • Figure 3 Flow chart of preparation of goat keratin by dialysis.
  • the filtrate is placed in a container with stirring for salting out, and ammonium sulfate aqueous solution is slowly added along the wall. Make the final concentration of ammonium sulfate 25%.
  • the protein will precipitate out.
  • filter, and the precipitate will be the crude goat keratin extract; add 400ml of pure water to the crude goat keratin extract.
  • the content of protein Q0VD04 was 2.50%; the content of Q14818 was 4.16%; the content of A5PJJ1 was 4.24%; the content of A3KN26 was 11.20%; the content of F1MC11 was 10.96%
  • the content of G3X7V4 is 1.70%; the content of M0QVY0 is 3.89%; the content of Q08D91 is 5.26%; the content of F1N362 is 4.60%; the content of E1B898 is 18.39%; the content of F1M198 is 6.42%; the content of Q5XQN5 is 9.66%. See Figure 1 for the flow chart of extraction and purification.
  • the protein is precipitated.
  • filter, and the precipitate is the crude goat keratin extract;
  • the crude goat keratin extract is suspended in 2000ml pure water.
  • the type and content of protein were determined for the obtained goat keratin.
  • the content of protein Q0VD04 was 3.54%; the content of Q14818 was 4.83%; the content of A5PJJ1 was 1.10%; the content of A3KN26 was 2.62%; the content of F1MC11 was 4.77%
  • the content of G3X7V4 is 1.01%; the content of M0QVY0 is 3.40%; the content of Q08D91 is 3.19%; the content of F1N362 is 3.86%; the content of E1B898 is 36.09%; the content of F1M198 is 10.10%; the content of Q5XQN5 is 8.73%. See Figure 1 for the flow chart of extraction and purification.
  • the type and content of protein were determined on the obtained goat keratin.
  • the content of protein Q0VD04 was 9.39%; the content of Q14818 was 3.94%; the content of A5PJJ1 was 3.00%; the content of A3KN26 was 7.79%; the content of F1MC11 was 3.46%
  • the content of G3X7V4 is 1.58%; the content of M0QVY0 is 4.98%; the content of Q08D91 is 6.74%; the content of F1N362 is 8.93%; the content of E1B898 is 21.44%; the content of F1M198 is 6.29%; the content of Q5XQN5 is 11.30%. See Figure 1 for the flow chart of extraction and purification.
  • 3L of the filtrate is concentrated and purified by an ultrafiltration system with a ceramic membrane core pore size of 5nm.
  • the liquid is 1L, add 1L of water to dilute and continue ultrafiltration purification.
  • the permeate is 1L, add 1L of water to continue ultrafiltration purification.
  • the permeate is discarded and the reflux liquid is recovered , Freeze-drying, that is, goat keratin is obtained. See Figure 2 for the flow chart of extraction and purification.
  • the filtrate is placed in a dialysis bag with a molecular weight cut-off of 3500Da.
  • the dialysis solvent ratio is 1:50.
  • the water was changed once every 10 hours, and after 8 times of water changes, the goat keratin retentate in the dialysis bag was recovered and freeze-dried to obtain goat keratin.
  • the type and content of protein were determined for the obtained goat keratin.
  • the content of protein Q0VD04 was 2.49%; the content of Q14818 was 4.99%; the content of A5PJJ1 was 2.48%; the content of A3KN26 was 2.71%; the content of F1MC11 was 4.35%
  • the content of G3X7V4 is 1.06%; the content of M0QVY0 is 3.60%; the content of Q08D91 is 3.20%; the content of F1N362 is 4.50%; the content of E1B898 is 35.38%; the content of F1M198 is 14.43%; the content of Q5XQN5 is 8.73%. See Figure 3 for the flow chart of extraction and purification.
  • the dialysis solvent ratio is 1:50, and the water is changed every 10h. After changing the water 8 times once, the goat keratin retentate in the dialysis bag is recovered and freeze-dried to obtain goat keratin. See Figure 3 for the flow chart of extraction and purification.
  • the dialysis solvent ratio is 1:50, and the water is changed every 10h. After changing the water 8 times, recover the goat keratin retentate in the dialysis bag and freeze-dry to obtain goat keratin. See Figure 3 for the flow chart of extraction and purification.
  • the dialysis solvent ratio is 1:50, and the water is changed every 10h. After changing the water 8 times, recover the goat keratin retentate in the dialysis bag and freeze-dry to obtain goat keratin. See Figure 3 for the flow chart of extraction and purification.
  • the dialysis solvent ratio is 1:50, and the water is changed every 10h. After changing the water 8 times, recover the goat keratin retentate in the dialysis bag and freeze-dry to obtain goat keratin. See Figure 3 for the flow chart of extraction and purification.
  • Drugs and reagents lipopolysaccharide, aspirin, different batches of goat keratin (S-1: Example 1 of the preparation method of goat keratin; S-2: Example 2 of the preparation method of goat keratin; S-3 : Example 3 of the preparation method of goat keratin; S-4: Example 4 of the preparation method of goat keratin; S-5: Example 5 of the preparation method of goat keratin)
  • Experimental method After the experimental animals are adapted to the experimental environment (temperature 22°C ⁇ 2°C, relative humidity 50% ⁇ 2%) for 1 day, they are pre-adapted to the operation of measuring rectal temperature at 8:00 and 15:00 respectively, and are forbidden for 12 hours before the experiment. Eat water and let the animals empty their feces before measuring the rectal temperature. Before each temperature measurement, apply petroleum jelly to the probe of the electronic thermometer and insert it into the rat rectum 2cm (it can be marked at 2cm to ensure that the insertion depth is the same).
  • mice normal control group; lipopolysaccharide fever model group; aspirin: 300mg/kg group; goat keratin samples: 10mg/kg group, 50mg/kg group.
  • Data statistics According to the body temperature measured at each time point on the day of the experiment, calculate the mean, standard deviation and standard error of the body temperature of each group of rats. Use TTEST to compare the data of each group between groups. P ⁇ 0.05 is considered as a significant difference.
  • the positive tool drug aspirin group can effectively inhibit the increase in body temperature of model rats at 2h, 4h, 6h and 8h after modeling. Compared with the model group, P ⁇ 0.05, there is a statistical difference, and the positive tool drug performance is relatively stable.
  • Example 2 Study on the effect of goat keratin on acetic acid writhing in ICR mice
  • Drugs and reagents aspirin, physiological saline, glacial acetic acid, different batches of goat keratin (S-1: Example 1 of the preparation method of goat keratin; S-2: Example 2 of the preparation method of goat keratin; S-3: Example 3 of the preparation method of goat keratin; S-4: Example 4 of the preparation method of goat keratin; S-5: Example 5 of the preparation method of goat keratin;)
  • mice After 1 day of acclimatization to the environment, the experimental animals were orally given aspirin 300mg/kg, goat keratin samples 50mg/kg, 200mg/kg, and the administration volume was 10ml/kg 1 hour in advance; then 0.6% acetic acid solution was injected into the abdominal cavity and observed The incubation period (seconds) and frequency of writhing in the animal within 15 minutes.
  • Experimental groups model group; aspirin: 300mg/kg group; goat keratin samples: 50mg/kg group, 200mg/kg group.
  • the aspirin 300mg/kg group can significantly delay the writhing latency and reduce the number of writhing times, and has a certain analgesic effect. Compared with the model group, P ⁇ 0.05, which is statistically significant.
  • the S-2 50mg/kg group showed the effect of reducing the number of writhing times in mice, and compared with the model group, P ⁇ 0.05, which was statistically significant; the S-5 50mg/kg group showed certain performance The degree delayed the latency of writhing in model animals and reduced the number of writhing, but compared with the model group, P>0.05, which was not statistically significant.
  • Drugs and reagents pilocarpine hydrochloride, diazepam tablets, retigabine, different batches of goat keratin (S-1: preparation method of goat keratin in Example 1; S-2: preparation of goat keratin) Method Example 2; S-3: Goat Keratin Preparation Method Example 3; S-4: Goat Keratin Preparation Method Example 4; S-5: Goat Keratin Preparation Method Example 5)
  • Experimental method model preparation and administration, once a day before the model is made, the drug is administered once in the afternoon, and the test drug is intraperitoneally injected 1 hour after the model is made. The positive drug is given 20 minutes before the model is made. The medicine is sufficient once.
  • seizure grade refers to Racine grading standard: grade 0: no reaction; grade I: manifested as facial muscles or It is the twitching of the corners of the mouth; grade II: you can nod; grade III: one limb twitch; grade IV: rigidity or whole body twitch; V grade: general epilepsy (general tonic seizure).
  • Experimental groups model group; retigabine: 60mg/kg group; diazepam tablets: 2mg/kg group; goat keratin samples: 50mg/kg group, 200mg/kg group.
  • grade IV attack rate in the model group was 80%. Seven out of 100 mice died. Positive drugs can inhibit the rate of grade IV epilepsy seizures, and significantly prolong the incubation period of grade III and grade IV epileptic seizures in mice. It shows that the model is successful.
  • the S-2-200mg/kg group has statistical difference compared with the model group.
  • the kg group, S-3-200mg/kg group, S-5-200mg/kg group and the model group have statistical differences.
  • epilepsy grade IV seizure latency there was no statistical difference between each dose group and the model group.
  • Example 4 Study on the effect of goat keratin sample on the expectorant effect of phenol red excretion method in mice
  • Drugs and reagents Mucosultan (ambroxol hydrochloride tablets), phenol red, sodium bicarbonate, different batches of goat keratin (S-1: Example 1 of the preparation method of goat keratin; S-2: goat horn) Keratin Preparation Method Example 2; S-3: Goat Keratin Preparation Method Example 3; S-4: Goat Keratin Preparation Method Example 4; S-5: Goat Keratin Preparation Method Example 5;)
  • mice were fasted and watered 16h before the experiment. Mucosultan and different doses of goat keratin drugs were given orally in groups (administration volume 10ml/kg). The solvent control group was given the same volume of distilled water. After 1 hour, 2.5% phenol red solution was injected intraperitoneally. After 30 minutes, the mice were sacrificed by necking. Take the trachea from below the thyroid cartilage to the branch of the trachea, put the trachea into 3ml of 5% NaHCO 3 solution and let it stand for 3h, take 1ml of supernatant, centrifuge at 3000rpm for 5min, measure and record the absorbance at 546nm. According to the standard curve of phenol red, the excretion of phenol red was calculated.
  • Example 5 Study on the antitussive effect of goat keratin sample on mice cough induced by ammonia water
  • Drugs and reagents dextromethorphan hydrobromide, ammonia, 0.2% CMC-Na, different batches of goat keratin (S-1: Example 1 of the preparation method of goat keratin; S-2: goat keratin) S-3: goat keratin preparation method embodiment 3; S-4: goat keratin preparation method embodiment 4; S-5: goat keratin preparation method embodiment 5;)
  • mice Orally administer dextromethorphan and different doses of goat keratin drugs (administration volume 10ml/kg) in groups.
  • the solvent control group was given the same volume of distilled water. After 1 hour, it was placed in a sealed box and atomized at a concentration of 10%. Ammonia water for 10 seconds, and then observe and record the incubation period of cough in mice and the number of coughs within 2 min.
  • the incubation period of cough refers to the number of seconds from the start of the atomization of ammonia to the occurrence of cough.
  • the performance of cough in mice is based on contraction of the abdominal muscles (breast reduction) and opening of the mouth at the same time. Calculate the mean and standard error of each group of data, use TTEST to compare the model group with other groups, and P ⁇ 0.05 is considered as a significant difference.
  • the dextromethorphan group has a significant improvement in the incubation period and the number of coughs, P ⁇ 0.05, which is statistically significant.
  • Example 6 Study on the efficacy of goat keratin samples on the yeast-induced fever model in SD rats
  • Experimental method Preparation of experimental animals: After the experimental animals are adapted to the experimental environment (temperature 22°C ⁇ 2°C, relative humidity 50% ⁇ 2%) for 1 day, they will be pre-adapted to measure rectal temperature at 8:00 and 15:00 respectively. Operation, fasting for 12 hours before the experiment, without water, let the animals empty the feces before measuring the rectal temperature. Before each temperature measurement, apply petroleum jelly to the probe of the electronic thermometer and insert it into the rat rectum 2cm (can be marked at 2cm to ensure the same depth of insertion each time), and record the body temperature after the reading is stable.
  • Subcutaneous injection of dry yeast to replicate the rat fever model measure the rat's body temperature before modeling, and select qualified rats with a body temperature of 36.2°C-37.3°C, and randomly group them into groups of 8 rats.
  • After oral administration of aspirin and different doses of goat keratin samples immediately subcutaneously injected 20% yeast suspension (10ml/kg), and the normal control group was injected with an equal volume of normal saline. After 2 hours, the rats' body temperature was monitored, every 2 hours thereafter. The body temperature was monitored once for a total of 8 hours.
  • mice fever model group normal control group
  • aspirin 300mg/kg group
  • goat keratin samples 10mg/kg group, 50mg/kg group.
  • Data statistics According to the body temperature measured at each time point on the day of the experiment, calculate the mean, standard deviation and standard error of the body temperature of each group of rats. Use TTEST to compare the data of each group between groups. P ⁇ 0.05 is considered as a significant difference.
  • the body temperature of rats in the model group increased significantly at 2h, 4h, 6h, and 8h after modeling. Compared with the normal group, P ⁇ 0.05, which was statistically different. The model was successfully established and stable and reliable.
  • the positive tool drug aspirin group can effectively inhibit the increase in body temperature of the model rats at 4h, 6h and 8h after modeling. Compared with the model group, P ⁇ 0.05, which is statistically significant.
  • the positive tool drug aspirin is stable.
  • the goat keratin samples can inhibit the body temperature rise of model rats to varying degrees after modeling, including S-2 50mg/kg group, S-3 10mg/kg group, S-4 10mg/kg group, S- 4
  • the 50mg/kg group and the S-5 10mg/kg group have a stronger effect. They can inhibit the increase in body temperature of model rats at most time points after modeling, and compared with the model group, P ⁇ 0.05, which is statistically significant.

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Abstract

公开了山羊角角蛋白、其制备方法、包含该山羊角角蛋白的药物组合物以及该山羊角角蛋白和该药物组合物在制备解热、镇痛、抗惊厥、镇咳、祛痰、降血压、抗炎、抗病毒药物中的用途，其中山羊角角蛋白通过去塞、干燥、粉碎、提取和纯化制备。

Description

一种山羊角角蛋白、其制备方法、其药物组合物及用途 技术领域
本发明属于医药技术领域，涉及一种山羊角角蛋白，其制备方法，含有该角蛋白的药物组合物，以及该角蛋白和所述药物组合物在制备解热、镇痛、抗惊厥、镇咳、祛痰、降血压、抗炎、抗病毒药物中的应用。
背景技术
山羊角为牛科(Bovidae)动物山羊属(Capra)青羊(Naemorhedus goral Hardwicke)、北山羊(Capra ibex Linnaeus)的角，性寒、咸，归心、肝经。功能主治在《本草新编》中描述为专活死血，磨山羊角一分，入酒中送下；在《医林纂要》中描述为功用近羚羊角；在《吉林中草药》中描述为镇静，退热，明目，止血，治小儿惊痫，头痛，产后腹痛，经痛，表明山羊角具有明显的药用价值。现代药理学研究，发现山羊角具有解热、镇静、抗惊厥、镇痛、抗病毒等作用，其主要的功效成分为角蛋白类物质。通过文献史料及现代药学研究，发现山羊角在功效应用上都类似于濒危动物药羚羊角，在《中国药典》2015年版一部中药成方制剂中将石斛夜光丸、金振口服液，北京同仁堂股份有限公司生产的卫生宝丸、同仁牛黄清心丸、羚羊清肺丸中的羚羊角均被山羊角替代，表明对于山羊角替代羚羊角的应用越来越得到允许和认可，但是山羊角用量与羚羊角相比有明显的差异，因此对于山羊角的研究，尤其是对于起主要作用的角蛋白类成分的研究更值得深入的探索和开发，以保证山羊角在临床应用中的安全性、方便性、资源可持续性。在此基础上，我们对山羊角角蛋白进行提取纯化，制备出山羊角角蛋白，为后续的开发利用奠定基础。
发明内容
本发明解决的技术问题是提供一类山羊角角蛋白、其制备方法， 药物组合物和应用。
为解决本发明的技术问题，本发明采取如下的技术方案:
本发明技术方案的第一方面是提供了一种山羊角角蛋白的制备方法，其特征在于，所述的制备方法如下:
(1)称取山羊角并用溶剂提取，即得山羊角角蛋白提取液。
优选的山羊角原药材是去塞、洗净后的山羊角。最优选的山羊角经干燥并适当的粉碎，以增大与溶剂的接触面积，提高提取效率。
山羊角细粉首先需冷浸提取，冷浸提取的溶剂为水，提取时溶剂的使用量为溶剂体积:山羊角细粉＝3～10:1(升/千克)，优选为5～8:1，更优选为5～6:1；提取的次数为2～5次，优选为3次；提取每次的时间为2～6h，优选为3～5h；提取可以在静态或动态下进行，优选在动态条件下，例如搅拌。冷浸提取后，离心，合并提取液，回收药渣备用。
冷浸提取后的药渣再经过还原提取，还原提取的溶剂为无机盐的水溶液，提取时溶剂的使用量为溶剂体积:冷浸提取后药渣的量＝15～100:1(升/千克)，优选为30～70:1，更优选为40～50:1；使用尿素的量:冷浸提取后药渣的量＝10～25:1(千克/千克)，优选为8～20:1，更优选为12～15:1；使用亚硫酸氢钠的量:冷浸提取后药渣的量＝1～5:1(千克/千克)，优选为2～4:1，更优选为2～3:1；使用十二烷基硫酸钠的量:冷浸提取后药渣的量＝0.25～2:1(千克/千克)，优选为0.5～1.5:1，更优选为0.75～1:1；使用蛋白酶抑制剂的量:冷浸提取后药渣的量＝0～2:1(升/千克)，优选为0～0.5:1，更优选为0～0.1:1；提取的温度为25～85℃，优选为50～80℃，更优选为65～75℃；提取的时间为3～24h，优选为6～18h，更优选为10～13h；提取可以在静态或动态下进行，优选在动态条件下，例如搅拌或者超声提取。还原提取后，离心，过滤，弃去药渣，即得山羊角角蛋白提取液。
(2)为了进一步得到山羊角角蛋白，步骤(1)中得到的山羊角角蛋白提取液需要除杂的步骤。
选用的除杂步骤，其具体包括：山羊角角蛋白提取液中，加入无机盐进行除杂，得到除杂后的山羊角角蛋白提取液。
其中，选用的无机盐为氯化钾。使用氯化钾的量:十二烷基硫酸钠的量＝1～3:1(千克/千克)，优选为2:1；除杂的温度为15～25℃，优选为18～22℃；除杂可以在静态或动态下进行，优选在动态条件下，例如搅拌。除杂后，离心，过滤，弃去沉淀，即得除杂后的山羊角角蛋白提取液。
(3)为进一步获得山羊角角蛋白组分，步骤(2)中制备的除杂后的山羊角角蛋白提取液需要浓缩、纯化的步骤。浓缩、纯化的步骤包括无机盐盐析的方法，陶瓷膜超滤系统超滤的方法及透析的方法。
其中无机盐盐析的方法中，选用的无机盐为硫酸铵、硫酸钠、氯化镁、硫酸镁、硫酸铝、硝酸铵、氯化铵、氯化钠或它们的水溶液，优选为硫酸铵的水溶液。加入无机盐使其终浓度达到10～50％，优选为20～40％，更优选为25～30％；山羊角角蛋白粗提物加入纯水再次盐析，使用纯水的体积:山羊角细粉＝10～30:1(升/千克)，优选为20:1；盐析次数为1～3次，优选为1次；山羊角角蛋白粗提物加入纯水清洗除杂，使用纯水的体积:山羊角细粉＝10～30:1(升/千克)，优选为20:1；山羊角角蛋白粗提物中加入纯水清洗的次数为2～5次，优选为3～5次。盐析，清洗除杂后得到的沉淀，冷冻干燥，即为山羊角角蛋白。
陶瓷膜超滤系统超滤的方法中，选用的陶瓷膜芯的孔径大小为5-10nm；除杂后的山羊角角蛋白提取液浓缩液体积:陶瓷膜超滤系统的死体积＝1～3:1(升/升)，优选为1～2:1；浓缩液加入等体积的纯水循环纯化的次数为5～15次，优选为8～12次；超滤纯化后，回收超滤系统中的纯化液，冷冻干燥，即得山羊角角蛋白。
透析的方法中，透析袋的截留分子量为3500-14000Da，优选为3500-5000Da；除杂后山羊角角蛋白提取液的体积:纯水的体积＝1:20～60(升/升)，优选为1:40～50；换水的间隔时间为6～12h，优选为8～10h；换水次数为5～10次，优选为6～8次；透析纯化后，回收透析袋中的截留液，冷冻干燥，即得山羊角角蛋白。
本发明技术方案的第二方面是提供第一方面所述的制备方法制备得到的山羊角角蛋白。进一步的，对得到的山羊角角蛋白，测定其种类及含量，其中蛋白质Q0VD04的含量为0.00～20.00％；Q14818的含 量为0.00～20.00％；A5PJJ1的含量为0.00～20.00％；F1MXG6的含量为0.00～20.00％；F1MC11的含量为0.00～20.00％；G3X7V4的含量为0.00～20.00％；M0QVY0的含量为0.00～20.00％；Q08D91的含量为0.00～20.00％；A0A140T867的含量为0.00～30.00％；F1N362的含量为0.00～20.00％；E1B898的含量为0.00～50.00％；F1M198的含量为0.00～30.00％；Q5XQN5的含量为0.00～25.00％。
进一步的，对得到的山羊角角蛋白，测定其种类及含量，其中蛋白质Q0VD04的含量为0.00～30.00％；Q14818的含量为0.00～20.00％；A5PJJ1的含量为0.00～40.00％；A3KN26的含量为0.00～50.00％；F1MC11的含量为0.00～20.00％；G3X7V4的含量为0.00～20.00％；M0QVY0的含量为0.00～20.00％；Q08D91的含量为0.00～50.00％；F1N362的含量为0.00～20.00％；E1B898的含量为0.00～50.00％；F1M198的含量为0.00～30.00％；Q5XQN5的含量为0.00～20.00％。
本发明技术方案的第三方面是提供了一种药物组合物，所述的组合物包含作为有效成分的第二方面所述的山羊角角蛋白及药学上可接受的载体或赋形剂。
本发明所述的药物组合物可根据本领域公知的方法制备。用于此目的时，如果需要，可将本发明山羊角角蛋白与一种或多种固体或液体药物赋形剂和/或辅剂结合，制成可作为人药或兽药使用的适当的施用形式或剂量形式。
本发明山羊角角蛋白或含有它的药物组合物可以单位剂量形式给药，给药途径可为肠道或非肠道，如口服、肌肉、皮下、鼻腔、口腔粘膜、眼、肺、皮肤、阴道、腹膜、直肠等，优选口服给药。
给药剂型可以是液体剂型、固体剂型或半固体剂型。液体剂型可以是溶液剂(包括真溶液和胶体溶液)、乳剂(包括水包油型、油包水型和复乳)、混悬剂、注射剂(包括水针剂、粉针剂和输液)、滴眼剂、滴鼻剂、洗剂和搽剂等。固体剂型可以是片剂(包括普通片、肠溶片、含片、分散片、咀嚼片、泡腾片、口腔崩解片)、胶囊剂(包括硬胶囊、软胶囊、肠溶胶囊)、颗粒剂、散剂、微丸、滴丸、栓剂、膜剂、贴片、气(粉)雾剂、喷雾剂等；半固体剂型可以是软膏剂、 凝胶剂、糊剂等。
本发明山羊角角蛋白可以制成普通制剂、也可以是缓释制剂、控释制剂、靶向制剂及各种微粒给药系统。
为了将单位给药剂型制成片剂，可以广泛使用本领域公知的各种赋形剂，包括稀释剂、黏合剂、润湿剂、崩解剂、润滑剂、助流剂。稀释剂可以是淀粉、糊精、蔗糖、葡萄糖、乳糖、甘露醇、山梨醇、木糖醇、微晶纤维素、硫酸钙、磷酸氢钙、碳酸钙等；湿润剂可以是水、乙醇、异丙醇等；粘合剂可以是淀粉浆、糊精、糖浆、蜂蜜、葡萄糖溶液、微晶纤维素、阿拉伯胶浆、明胶浆、羧甲基纤维素钠、甲基纤维素、羟丙基甲基纤维素、乙基纤维素、丙烯酸树脂、卡波姆、聚乙烯吡咯烷酮、聚乙二丙醇等；崩解剂可以是干淀粉、微晶纤维素、低取代羟丙基纤维素、交联聚乙烯吡咯烷酮、交联羧甲基纤维素钠、羧甲基淀粉钠、碳酸氢钠与构椽酸、碳酸钙、聚氧乙烯山梨糖醇脂肪酸酯、十二烷基磺酸钠；润滑剂和助流剂可以是滑石粉、二氧化硅、硬脂酸盐、酒石酸、液体石蜡、聚乙二醇等。
还可以将片剂进一步制成包衣片，例如糖包衣片、薄膜包衣片、肠溶包衣片，或双层片和多层片。
为了将给药单元制成丸剂，可以广泛使用本领域公知的各种载体。关于载体的例子是，例如稀释剂与吸收剂，如葡萄糖、乳糖、淀粉、可可脂、氢化植物油、聚乙烯吡咯烷酮、月桂酸聚乙二醇甘油酯、高岭土、滑石粉等；粘合剂，如阿拉伯胶、黄菩胶、明胶、乙醇、蜂蜜、液糖、米糊或面糊等；崩解剂，如琼脂粉、干燥淀粉、海藻酸盐、十二烷基磺酸钠、甲基纤维素、乙基纤维素等。
为了将给药单元制成栓剂，可以广泛使用本领域公知的各种载体。关于载体的例子是，例如聚乙二醇、卵磷脂、可可脂、高级醇、高级醇的酯、明胶、半合成甘油酯等。
为了将给药单元制成胶囊，将山羊角角蛋白或含有山羊角角蛋白的药物组合物与上述的各种载体混合，并将由此得到的混合物置于硬的明胶胶囊或软胶囊中。也可将有效成分本发明山羊角角蛋白制成微囊剂，混悬于水性介质中形成混悬剂，亦可装入硬胶囊中应用。
例如，将本发明山羊角角蛋白制成溶液剂、混悬剂溶液剂、乳剂、冻干粉针剂，这种制剂可以是含水或非水的，可含一种和/或多种药效学上可接受的载体、稀释剂、粘合剂、润滑剂、防腐剂、表面活性剂或分散剂。如稀释剂可选自水、乙醇、聚乙二醇、l,3-丙二醇、乙氧基化的异硬脂醇、多氧化的异硬脂醇、聚氧乙烯山梨醇脂肪酸酯等。另外，为了制备等渗注射液，可以向注射用制剂中添加适量的氯化钠、葡萄糖或甘油，此外，还可以添加常规的助溶剂、缓冲剂、pH调节剂等。这些辅料是本领域常用的。
此外，如需要，也可以向药物制剂中添加着色剂、防腐剂、香料、矫味剂、甜味剂或其它材料。
为达到用药目的，增强治疗效果，本发明的山羊角角蛋白或药物组合物可用任何公知的给药方法给药。
本发明山羊角角蛋白药物组合物的给药剂量取决于许多因素，例如所要预防或治疗疾病的性质和严重程度，患者或动物的性别、年龄、体重、性格及个体反应，给药途径、给药次数、治疗目的，因此本发明的治疗剂量可以有大范围的变化。一般来讲，本发明中药学成分的使用剂量是本领域技术人员公知的。可以根据本发明山羊角角蛋白组合物中最后的制剂中所含有的实际药物数量，加以适当的调整，以达到其治疗有效量的要求，完成本发明的预防或治疗目的。本发明山羊角角蛋白的每天的合适剂量范围:本发明的山羊角角蛋白的用量为0.01～500mg/Kg体重，优选为0.5～100mg/Kg体重，更优选为1～50mg/Kg体重，最优选为2～30mg/Kg体重。上述剂量可以单一剂量形式或分成几个，例如二、三或四个剂量形式给药，这取决于给药医生的临床经验以及包括运用其它治疗手段的给药方案。每一种治疗所需总剂量可分成多次或按一次剂量给药。本发明的山羊角角蛋白或药物组合物可单独服用，或与其他治疗药物或对症药物合并使用并调整剂量。
本发明技术方案的第四方面是提供一种山羊角角蛋白在制备解热、镇痛、抗惊厥、镇咳、祛痰、降血压、抗炎、抗病毒药物中的应用。
本发明技术方案的第四方面还提供第二方面所述的山羊角角蛋白 和第三方面所述的药物组合物在制备解热、镇痛、抗惊厥、镇咳、祛痰、降血压、抗炎、抗病毒药物中的应用。
本发明具有的技术优势为:
1、为纯天然成分。
2、动物体内模型研究表明其具有明显的解热、镇痛、抗惊厥、镇咳、祛痰、降血压、抗炎、抗病毒活性。
3、药材易得，提取工艺简单，适合大规模工业化生产。
附图说明
图1：盐析法山羊角角蛋白的制备流程图。
图2：超滤法山羊角角蛋白的制备流程图。
图3：透析法山羊角角蛋白的制备流程图。
具体实施方式
下面的实施例用来进一步说明本发明，但这并不意味着对本发明的任何限制。
实施例1：山羊角角蛋白的制备方法
称量去塞、清洗、晾干后的山羊角22g，粉碎，过100目筛，用6倍量纯水室温搅拌提取3次，提取时间分别为5h，4h和3h，提取后的药渣20g中加入纯水1000ml，尿素300g，亚硫酸氢钠40g，十二烷基硫酸钠15g，蛋白酶抑制剂10ml，75℃下搅拌提取12h，离心，过滤，即得山羊角角蛋白提取液，提取液在搅拌的情况下加入30g的氯化钾固体，待产生白色沉淀呈混悬液为止，静置，离心，过滤，滤液置于带搅拌的容器中进行盐析，沿壁缓慢加入硫酸铵水溶液，使硫酸铵的终浓度为25％，盐析过程中，蛋白析出，待盐析完全后，过滤，沉淀即为山羊角角蛋白粗提物；山羊角角蛋白粗提物中加入400ml纯水混悬，再次沿壁缓慢加入硫酸铵水溶液，使硫酸铵的终浓度仍为25％，进行第二次盐析，过滤，沉淀即为山羊角角蛋白粗提物；山羊角角蛋白粗提物中加入400ml纯水混悬，搅拌清洗，静置，过滤，重复上述操作3次后，沉淀冷冻干燥，即得山羊角角蛋白。对所得的山羊角角蛋白测定蛋白质的种类及含量，其中蛋白质Q0VD04的含量为2.50％；Q14818的含量为4.16％；A5PJJ1的含量为4.24％；A3KN26的含量为 11.20％；F1MC11的含量为10.96％；G3X7V4的含量为1.70％；M0QVY0的含量为3.89％；Q08D91的含量为5.26％；F1N362的含量为4.60％；E1B898的含量为18.39％；F1M198的含量为6.42％；Q5XQN5的含量为9.66％。提取纯化的流程图见附图1。
实施例2：山羊角角蛋白的制备方法
称量去塞、清洗、晾干后的山羊角105g，粉碎，过100目筛，用6倍量纯水室温搅拌提取3次，提取时间分别为5h，4h和3h，提取后的药渣100g中加入纯水5000ml，尿素1500g，亚硫酸氢钠200g，十二烷基硫酸钠75g，蛋白酶抑制剂5ml，75℃下搅拌提取12h，离心，即得山羊角角蛋白提取液，提取液在搅拌的情况下加入150g的氯化钾固体，待产生白色沉淀呈混悬液为止，静置，离心，过滤，滤液置于带搅拌的容器中进行盐析，沿壁缓慢加入硫酸铵水溶液，使硫酸铵的终浓度为25％，盐析过程中，蛋白析出，待盐析完全后，过滤，沉淀即为山羊角角蛋白粗提物；山羊角角蛋白粗提物中加入2000ml纯水混悬，再次沿壁缓慢加入硫酸铵水溶液，使硫酸铵的终浓度仍为25％，进行第二次盐析，过滤，沉淀即为山羊角角蛋白粗提物；山羊角角蛋白粗提物中加入2000ml纯水混悬，搅拌清洗，静置，过滤，重复上述操作3次后，沉淀冷冻干燥，即得山羊角角蛋白。对所得的山羊角角蛋白测定蛋白质的种类及含量，其中蛋白质Q0VD04的含量为3.54％；Q14818的含量为4.83％；A5PJJ1的含量为1.10％；A3KN26的含量为2.62％；F1MC11的含量为4.77％；G3X7V4的含量为1.01％；M0QVY0的含量为3.40％；Q08D91的含量为3.19％；F1N362的含量为3.86％；E1B898的含量为36.09％；F1M198的含量为10.10％；Q5XQN5的含量为8.73％。提取纯化的流程图见附图1。
实施例3：山羊角角蛋白的制备方法
称量去塞、清洗、晾干后的山羊角105g，粉碎，过100目筛，用6倍量纯水室温搅拌提取3次，提取时间分别为5h，4h和3h，提取后的药渣100g中加入纯水5000ml，尿素1500g，亚硫酸氢钠200g，十二烷基硫酸钠75g，75℃下搅拌提取12h，离心，即得山羊角角蛋白提取液，提取液在搅拌的情况下加入150g的氯化钾固体，待产生白色沉淀呈混 悬液为止，静置，离心，过滤，滤液置于带搅拌的容器中进行盐析，沿壁缓慢加入硫酸铵水溶液，使硫酸铵的终浓度为25％，盐析过程中，蛋白析出，待盐析完全后，过滤，沉淀即为山羊角角蛋白粗提物；山羊角角蛋白粗提物中加入2000ml纯水混悬，再次沿壁缓慢加入饱和硫酸铵水溶液，使硫酸铵的终浓度仍为25％，进行第二次盐析，过滤，沉淀即为山羊角角蛋白粗提物；山羊角角蛋白粗提物中加入2000ml纯水混悬，搅拌清洗，静置，过滤，重复上述操作3次后，沉淀冷冻干燥，即得山羊角角蛋白。对所得的山羊角角蛋白测定蛋白质的种类及含量，其中蛋白质Q0VD04的含量为9.39％；Q14818的含量为3.94％；A5PJJ1的含量为3.00％；A3KN26的含量为7.79％；F1MC11的含量为3.46％；G3X7V4的含量为1.58％；M0QVY0的含量为4.98％；Q08D91的含量为6.74％；F1N362的含量为8.93％；E1B898的含量为21.44％；F1M198的含量为6.29％；Q5XQN5的含量为11.30％。提取纯化的流程图见附图1。
实施例4：山羊角角蛋白的制备方法
称量去塞、清洗、晾干后的山羊角52g，粉碎，过100目筛，用6倍量纯水室温搅拌提取3次，提取时间分别为5h，4h和3h，提取后的药渣50g中加入纯水2500ml，尿素750g，亚硫酸氢钠100g，十二烷基硫酸钠37.5g，酶抑制剂2.5ml，75℃下搅拌提取12h，离心，即得山羊角角蛋白提取液，提取液在搅拌的情况下加入75g的氯化钾粉末，待产生白色沉淀呈混悬液为止，静置，离心，过滤，滤液3L用陶瓷膜芯孔径为5nm的超滤系统进行浓缩和纯化，待浓缩液为1L时，加入1L水稀释后继续超滤纯化，待透过液为1L时，再加入1L水继续超滤纯化，如此循环超滤纯化10次后，弃去透过液，回收回流液，冷冻干燥，即得山羊角角蛋白。提取纯化的流程图见附图2。
实施例5：山羊角角蛋白的制备方法
称量去塞、清洗、晾干后的山羊角22g，粉碎，过100目筛，用6倍量纯水室温搅拌提取3次，提取时间分别为5h，4h和3h，提取后的药渣20g中加入纯水1000ml，尿素300g，亚硫酸氢钠40g，十二烷基硫酸钠15g，蛋白酶抑制剂10ml，75℃下搅拌提取12h，离心，即得山 羊角角蛋白提取液，提取液在搅拌的情况下加入30g的氯化钾粉末，待产生白色沉淀呈混悬液为止，静置，离心，过滤，滤液置于截留分子量为3500Da的透析袋中，透析的溶剂比为1:50，每隔10h换水一次，换水8次后，回收透析袋中的山羊角角蛋白截留液，冷冻干燥，即得山羊角角蛋白。对所得的山羊角角蛋白测定蛋白质的种类及含量，其中蛋白质Q0VD04的含量为2.49％；Q14818的含量为4.99％；A5PJJ1的含量为2.48％；A3KN26的含量为2.71％；F1MC11的含量为4.35％；G3X7V4的含量为1.06％；M0QVY0的含量为3.60％；Q08D91的含量为3.20％；F1N362的含量为4.50％；E1B898的含量为35.38％；F1M198的含量为14.43％；Q5XQN5的含量为8.73％。提取纯化的流程图见附图3。
实施例6：山羊角角蛋白的制备方法
称量去塞、清洗、晾干后的山羊角12g，粉碎，过100目筛，用6倍量纯水室温搅拌提取3次，提取时间分别为5h，4h和3h，提取后的药渣10g中加入纯水250ml，尿素75g，亚硫酸氢钠20g，十二烷基硫酸钠3.75g，75℃下搅拌提取6h，离心，即得山羊角角蛋白提取液，提取液在搅拌的情况下加入7.5g氯化钾粉末，待产生白色沉淀呈混悬液为止，静置，离心，过滤，滤液置于截留分子量为3500Da的透析袋中，透析的溶剂比为1:50，每隔10h换水一次，换水8次后，回收透析袋中的山羊角角蛋白截留液，冷冻干燥，即得山羊角角蛋白。提取纯化的流程图见附图3。
实施例7：山羊角角蛋白的制备方法
称量去塞、清洗、晾干后的山羊角12g，粉碎，过100目筛，用6倍量纯水室温搅拌提取3次，提取时间分别为5h，4h和3h，提取后的药渣10g中加入纯水500ml，尿素150g，亚硫酸氢钠10g，十二烷基硫酸钠7.5g，75℃下搅拌提取12h，离心，即得山羊角角蛋白提取液，提取液在搅拌的情况下加入15g氯化钾粉末，待产生白色沉淀呈混悬液为止，静置，离心，过滤，滤液置于截留分子量为3500Da的透析袋中，透析的溶剂比为1:50，每隔10h换水一次，换水8次后，回收透析袋中的山羊角角蛋白截留液，冷冻干燥，即得山羊角角蛋白。提取纯化 的流程图见附图3。
实施例8：山羊角角蛋白的制备方法
称量去塞、清洗、晾干后的山羊角12g，粉碎，过100目筛，用6倍量纯水室温搅拌提取3次，提取时间分别为5h，4h和3h，提取后的药渣10g中加入纯水150ml，尿素45g，亚硫酸氢钠30g，十二烷基硫酸钠2.5g，75℃下搅拌提取24h，离心，即得山羊角角蛋白提取液，提取液在搅拌的情况下加入5g氯化钾粉末，待产生白色沉淀呈混悬液为止，静置，离心，过滤，滤液置于截留分子量为3500Da的透析袋中，透析的溶剂比为1:50，每隔10h换水一次，换水8次后，回收透析袋中的山羊角角蛋白截留液，冷冻干燥，即得山羊角角蛋白。提取纯化的流程图见附图3。
实施例9：山羊角角蛋白的制备方法
称量去塞、清洗、晾干后的山羊角12g，粉碎，过100目筛，用6倍量纯水室温搅拌提取3次，提取时间分别为5h，4h和3h，提取后的药渣10g中加入纯水150ml，尿素150g，亚硫酸氢钠20g，十二烷基硫酸钠7.5g，75℃下超声提取6h，离心，即得山羊角角蛋白提取液，提取液在搅拌的情况下加入15g氯化钾粉末，待产生白色沉淀呈混悬液为止，静置，离心，过滤，滤液置于截留分子量为3500Da的透析袋中，透析的溶剂比为1:50，每隔10h换水一次，换水8次后，回收透析袋中的山羊角角蛋白截留液，冷冻干燥，即得山羊角角蛋白。提取纯化的流程图见附图3。
药理实验
实施例1：山羊角角蛋白对LPS诱导SD大鼠发热模型的药效研究
动物：雄性SD大鼠
药品与试剂：脂多糖、阿司匹林、不同批次山羊角角蛋白(S-1：山羊角角蛋白的制备方法实施例1；S-2：山羊角角蛋白的制备方法实施例2；S-3：山羊角角蛋白的制备方法实施例3；S-4：山羊角角蛋白的制备方法实施例4；S-5：山羊角角蛋白的制备方法实施例5)
实验方法：实验动物在实验环境(温度22℃±2℃，相对湿度50％±2％)中适应1天后，分别于早8:00及15:00预适应测量肛温操作，实验前12h禁食不禁水，测定肛温前让动物排空粪便。每次测温前电子体温计探头涂抹凡士林，插入大鼠直肠2cm(可在2cm处标记，确保每次插入深度一致)，待读数稳定以后记录体温值；腹腔注射脂多糖复制大鼠发热模型：造模前测大鼠体温，筛选出体温在36.2℃-37.3℃的合格大鼠，随机分组，每组8只。口服给予阿司匹林及不同剂量山羊角角蛋白样品后立即腹腔注射脂多糖(20ug/kg、2ml/kg)，正常对照组腹腔注射等体积生理盐水，2h后开始监测大鼠体温，共监测8h。
实验分组：正常对照组；脂多糖发热模型组；阿司匹林：300mg/kg组；山羊角角蛋白样品：10mg/kg组、50mg/kg组。
数据统计：根据实验当天测量的各时间点体温值，计算各组大鼠体温均值、标准差及标准误，应用TTEST将各组数据进行组间比较，P<0.05认为有显著性差异。
实验结果(详见表1-1、1-2、1-3)：
1、腹腔注射20ug/kg脂多糖可成功诱导大鼠体温升高，模型组大鼠在造模2h、4h、6h、8h体温明显升高，与正常组比较，P<0.05，有统计学差异，模型稳定。
2、阳性工具药阿司匹林组在造模2h、4h、6h、8h均可以有效抑制模型大鼠体温升高，与模型组比较，P<0.05，有统计学差异，阳性工具药表现较稳定。
3、山羊角角蛋白样品S-2 50mg/kg组在造模后8h，S-3 10mg/kg在造模后4h及8h可明显降低模型大鼠体温，S-4 10mg/kg组，S-4 50mg/kg组在造模后2h及4h可明显降低模型大鼠体温，与模型组比较，P<0.05，有统计学差异。S-5 50mg/kg组在造模后2h显示一定程度降低模型大鼠体温作用趋势，但与模型组比较，P>0.05，无统计学差异。
表1-1 不同剂量山羊角角蛋白样品对LPS诱导大鼠发热模型的影响
Figure PCTCN2020119336-appb-000001
与模型组比较：#P<0.05，##P<0.01，###P<0.001；与正常对照组比较:***P<0.001
表1-2 不同剂量山羊角角蛋白样品对LPS诱导大鼠发热模型的影响
Figure PCTCN2020119336-appb-000002
与模型组比较：#P<0.05，##P<0.01，###P<0.001；与正常对照组比较:***P<0.001
表1-3 不同剂量山羊角角蛋白样品对LPS诱导大鼠发热模型的影响
Figure PCTCN2020119336-appb-000003
Figure PCTCN2020119336-appb-000004
与模型组比较：#P<0.05，##P<0.01，###P<0.001；与正常对照组比较:***P<0.001
实施例2：山羊角角蛋白对对ICR小鼠醋酸扭体的药效研究
动物：ICR小鼠雌性
药品与试剂：阿司匹林、生理盐水、冰醋酸、不同批次山羊角角蛋白(S-1：山羊角角蛋白的制备方法实施例1；S-2：山羊角角蛋白的制备方法实施例2；S-3：山羊角角蛋白的制备方法实施例3；S-4：山羊角角蛋白的制备方法实施例4；S-5：山羊角角蛋白的制备方法实施例5；)
实验方法：实验动物适应环境1天后，提前1h分别口服给予阿司匹林300mg/kg、山羊角角蛋白样品50mg/kg、200mg/kg，给药体积为10ml/kg；而后腹腔注射0.6％醋酸溶液，观察15分钟内动物出现扭体潜伏期(秒)及次数。
实验分组：模型组；阿司匹林：300mg/kg组；山羊角角蛋白样品：50mg/kg组、200mg/kg组。
数据统计：所有组别数据计算均值及标准误，应用TTEST，与模型组比较，P<0.05认为有统计学差异。
实验结果(详见表2-1、2-2、2-3)：
1、阿司匹林300mg/kg组可明显后延扭体潜伏期和减少扭体次数，具有一定的镇痛作用，与模型组比较，P<0.05，有统计学意义。
2、山羊角角蛋白样品中S-2 50mg/kg组表现出减少小鼠扭体次数作用，且与模型组比较，P<0.05，有统计学意义；S-5 50mg/kg组表现有一定程度后延模型动物扭体潜伏期并减少扭体次数，但与模型组比较，P>0.05，无统计学意义。
表2-1 不同剂量山羊角角蛋白样品对ICR小鼠醋酸扭体实验的影响
Figure PCTCN2020119336-appb-000005
Figure PCTCN2020119336-appb-000006
与模型组比较：*P<0.05，**P<0.01，***P<0.001
表2-2 不同剂量山羊角角蛋白样品对ICR小鼠醋酸扭体实验的影响
Figure PCTCN2020119336-appb-000007
与模型组比较：*P<0.05，**P<0.01，***P<0.001
表2-3 不同剂量山羊角角蛋白样品对ICR小鼠醋酸扭体实验的影响
Figure PCTCN2020119336-appb-000008
与模型组比较：*P<0.05，**P<0.01，***P<0.001
实施例3：山羊角角蛋白样品对盐酸毛果芸香碱(PLO)致小鼠癫痫的药效研究
动物：ICR小鼠雄性
药品与试剂：盐酸毛果芸香碱、地西泮片、瑞替加滨、不同批次山羊角角蛋白(S-1：山羊角角蛋白的制备方法实施例1；S-2：山羊角角蛋白的制备方法实施例2；S-3：山羊角角蛋白的制备方法实施例3；S-4：山羊角角蛋白的制备方法实施例4；S-5：山羊角角蛋白的制备方法实施例5)
实验方法：模型制备及给药，造模前1天下午给药一次，造模当天灌胃受试药后1h腹腔注射PLO-225mg/kg(造模剂)，阳性药在造模 前20min给药一次即可。注射PLO后持续观察30min；观察指标：①癫痫发作情况：Ⅱ级～Ⅳ级发作时间；②死亡时间；发作级别：参照Racine分级标准：0级：没有任何反应；I级：表现为面部肌肉或是嘴角的抽动；Ⅱ级：可以点头；Ⅲ级：一侧肢体抽搐；Ⅳ级：强直或全身肢体抽搐；V级：癫痫全面大发作(全身强直性惊厥发作)。
实验分组：模型组；瑞替加滨：60mg/kg组；地西泮片：2mg/kg组；山羊角角蛋白样品:50mg/kg组、200mg/kg组。
数据统计：统计实验中各组小鼠Ⅳ级发作及死亡例数；Ⅱ级、Ⅲ级及Ⅳ级潜伏期，未发作至Ⅳ级的小鼠潜伏期记作最大值1800s。例数统计用卡方检验统计。潜伏期计算均值及标准误，应用TTEST，将模型组与其他各组进行组间比，P<0.05认为有显著性差异。
实验结果(详见表3-1、3-2、3-3；表4-1、4-2、4-3)：
1、模型组Ⅳ级发作率为80％。100只小鼠有7只死亡。阳性药可以抑制癫痫Ⅳ级发作率，并显著延长小鼠Ⅲ级，Ⅳ级癫痫发作潜伏期。说明造模成功。
2、在癫痫Ⅱ级潜伏期比较中，S-2-200mg/kg组与模型组比较有统计学差异。在癫痫Ⅲ级潜伏期比较中，S-1-50mg/kg组，S-1-200mg/kg组，S-2-50mg/kg组，S-2-200mg/kg组，S-3-50mg/kg组，S-3-200mg/kg组，S-5-200mg/kg组与模型组比较均有统计学差异。在癫痫Ⅳ级发作潜伏期比较中，各剂量组与模型组比较均无统计学差异。
表3-1 山羊角角蛋白样品对PTZ致小鼠癫痫实验-例数统计
Figure PCTCN2020119336-appb-000009
Figure PCTCN2020119336-appb-000010
与模型组比较：*P<0.05，**P<0.01
表3-2 山羊角角蛋白样品对PTZ致小鼠癫痫实验-例数统计
Figure PCTCN2020119336-appb-000011
与模型组比较：*P<0.05，**P<0.01
表3-3 山羊角角蛋白样品对PTZ致小鼠癫痫实验-例数统计
Figure PCTCN2020119336-appb-000012
与模型组比较：*P<0.05，**P<0.01
表4-1 山羊角角蛋白样品对PTZ致小鼠癫痫实验-Ⅲ级及Ⅳ级癫痫发作潜伏期(mean±SEM)
Figure PCTCN2020119336-appb-000013
与模型组比较：*P<0.05，**P<0.01
表4-2 山羊角角蛋白样品对PTZ致小鼠癫痫实验-Ⅲ级及Ⅳ级癫痫发作潜伏期(mean±SEM)
Figure PCTCN2020119336-appb-000014
Figure PCTCN2020119336-appb-000015
与模型组比较：*P<0.05，**P<0.01
表4-3 山羊角角蛋白样品对PTZ致小鼠癫痫实验-Ⅲ级及Ⅳ级癫痫发作潜伏期(mean±SEM)
Figure PCTCN2020119336-appb-000016
与模型组比较：*P<0.05，**P<0.01
实施例4：山羊角角蛋白样品对小鼠酚红排泄法祛痰的药效研究
动物：ICR小鼠，雄性
药品与试剂：沐舒坦(盐酸氨溴索片)、苯酚红、碳酸氢钠、不同批次山羊角角蛋白(S-1：山羊角角蛋白的制备方法实施例1；S-2：山羊角角蛋白的制备方法实施例2；S-3：山羊角角蛋白的制备方法实施例3；S-4：山羊角角蛋白的制备方法实施例4；S-5：山羊角角蛋白的制备方法实施例5；)
实验方法：动物于实验前16h禁食不禁水。按分组口服给予沐舒坦及不同剂量山羊角角蛋白药物(给药体积10ml/kg)，溶剂对照组给予等量体积蒸馏水，1h后腹腔注射2.5％酚红溶液，30min后脱颈处死小鼠，取自甲状软骨下至气管分支前一段气管，将气管放入3ml5％NaHCO 3溶液静置3h，取上清1ml，3000rpm离心5min后，546nm处测量并记录吸光度。根据酚红的标准曲线计算出酚红的排泄量。
实验分组：溶剂对照组；沐舒坦：30mg/kg组；山羊角角蛋白样品：20mg/kg组、50mg/kg组。
数据统计：分别记录口服给药时间点，2.5％酚红溶液腹腔注射时间点，取气管时间点；酶标仪546nm处测量得到各组样本吸光度，根据酚红的标准曲线计算出酚红的排泄量。各组数据计算均值及标准误， 应用TTEST，将溶剂对照组与其他各组进行组间比，P<0.05认为有显著性差异。
实验结果(详见表5-1、5-2、5-3)：
1、沐舒坦30mg/kg组与溶剂对照组比较，酚红排泄量明显升高，P<0.05，具有统计学意义。
2、山羊角角蛋白样品S-1 20mg/kg组，S-1 50mg/kg组，S-2 20mg/kg组，S-2 50mg/kg组，S-320mg/kg组，S-3 50mg/kg组，S-5 20mg/kg组，S-5 50mg/kg组与溶剂对照组比较，酚红排泄量明显升高，P<0.05，具有统计学意义。
表5-1 口服山羊角角蛋白样品对小鼠酚红排泄法祛痰药效实验(X±SEM)
Figure PCTCN2020119336-appb-000017
与溶剂对照组比较：*P<0.05，与溶剂对照组比较：**P<0.01
表5-2 口服山羊角角蛋白样品对小鼠酚红排泄法祛痰药效实验(X±SEM)
Figure PCTCN2020119336-appb-000018
与溶剂对照组比较：*P<0.05，与溶剂对照组比较：**P<0.01
表5-3 口服山羊角角蛋白样品对小鼠酚红排泄法祛痰药效实验(X±SEM)
Figure PCTCN2020119336-appb-000019
Figure PCTCN2020119336-appb-000020
与溶剂对照组比较：*P<0.05，与溶剂对照组比较：**P<0.01
实施例5：山羊角角蛋白样品对小鼠氨水引咳法镇咳的药效研究
动物：ICR小鼠雄性
药品与试剂：氢溴酸右美沙芬、氨水、0.2％CMC-Na、不同批次山羊角角蛋白(S-1：山羊角角蛋白的制备方法实施例1；S-2：山羊角角蛋白的制备方法实施例2；S-3：山羊角角蛋白的制备方法实施例3；S-4：山羊角角蛋白的制备方法实施例4；S-5：山羊角角蛋白的制备方法实施例5；)
实验方法：按分组口服给予右美沙芬及不同剂量山羊角角蛋白药物(给药体积10ml/kg)，溶剂对照组给予等量体积蒸馏水，1h后放入密封盒内，通雾化10％浓度氨水10秒钟，然后观察和记录小鼠的咳嗽潜伏期和2min内咳嗽次数。
实验分组：溶剂对照组；右美沙芬：15mg/kg组；山羊角角蛋白样品：20mg/kg组、50mg/kg组。
数据统计：分别记录口服给药时间点，雾化实验时间点，小鼠的咳嗽潜伏期和2min内咳嗽次数。咳嗽潜伏期是指雾化氨水开始时间至发生咳嗽所需的秒数。小鼠咳嗽表现以其腹肌收缩(缩胸)，同时张大嘴为准。各组数据计算均值及标准误，应用TTEST，将模型组与其他各组进行组间比，P<0.05认为有显著性差异。
实验结果(详见表6-1、6-2、6-3)：
1、右美沙芬组与溶剂对照组在潜伏期和咳嗽次数上比较，有明显的改善作用，P<0.05，具有统计学意义。
2、山羊角角蛋白样品S-1 50mg/kg组、S-2 50mg/kg组，在潜伏期上与溶剂对照组比较，有明显的改善作用，P<0.05，具有统计学意义；S-150mg/kg组、S-2 50mg/kg组、S-3 20mg/kg组，S-5 50mg/kg组，在咳嗽次数上与溶剂对照组比较，有明显改善作用，P<0.05，具有统计学意义。
表6-1 口服山羊角角蛋白对小鼠氨水引咳法的镇咳药效实验(X±SEM)
Figure PCTCN2020119336-appb-000021
Figure PCTCN2020119336-appb-000022
与溶剂对照组比较： *P<0.05，与溶剂对照组比较： **P<0.01
表6-2 口服山羊角角蛋白对小鼠氨水引咳法的镇咳药效实验(X±SEM)
Figure PCTCN2020119336-appb-000023
与溶剂对照组比较： *P<0.05，与溶剂对照组比较： **P<0.01
表6-3 口服山羊角角蛋白对小鼠氨水引咳法的镇咳药效实验(X±SEM)
Figure PCTCN2020119336-appb-000024
与溶剂对照组比较： *P<0.05，与溶剂对照组比较： **P<0.01
实施例6：山羊角角蛋白样品对对酵母诱导SD大鼠发热模型的药效研究
动物：雄性SD大鼠
药品及试剂：酵母菌，阿司匹林，不同批次山羊角角蛋白(S-1：山羊角角蛋白的制备方法实施例1；S-2：山羊角角蛋白的制备方法实施例2；S-3：山羊角角蛋白的制备方法实施例3；S-4：山羊角角蛋白的制备方法实施例4；S-5：山羊角角蛋白的制备方法实施例5；)
实验方法：实验动物的准备：实验动物在实验环境(温度22℃±2℃， 相对湿度50％±2％)中适应1天后，分别于早8:00及15:00进行预适应测量肛温操作，实验前12h禁食不禁水，测定肛温前让动物排空粪便。每次测温前电子体温计探头涂抹凡士林，插入大鼠直肠2cm(可在2cm处标记，确保每次插入深度一致)，待读数稳定以后记录体温值。皮下注射干酵母复制大鼠发热模型：造模前测大鼠体温，筛选出体温在36.2℃-37.3℃的合格大鼠，随机分组，每组8只。口服给予阿司匹林、不同剂量山羊角角蛋白样品后立即皮下注射20％酵母菌混悬液(10ml/kg)，正常对照组皮下注射等体积生理盐水，2h后开始监测大鼠体温，此后每间隔2h监测体温1次，共监测8h。
实验分组：正常对照组；酵母菌发热模型组；阿司匹林：300mg/kg组；山羊角角蛋白样品：10mg/kg组、50mg/kg组。
数据统计：根据实验当天测量的各时间点体温值，计算各组大鼠体温均值、标准差及标准误，应用TTEST将各组数据进行组间比较，P<0.05认为有显著性差异。
实验结果(详见表7-1、7-2、7-3)：
1、模型组大鼠在造模2h、4h、6h、8h体温明显升高，与正常组比较，P<0.05，有统计学差异，模型建立成功且稳定可靠。
2、阳性工具药阿司匹林组在造模4h、6h、8h均可以有效抑制模型大鼠体温升高，与模型组比较，P<0.05，有统计学意义，阳性工具药阿司匹林表现稳定。
3、山羊角角蛋白样品在造模后均可不同程度抑制模型大鼠体温升高,其中S-2 50mg/kg组，S-3 10mg/kg组，S-4 10mg/kg组，S-4 50mg/kg组，S-5 10mg/kg组作用较强，在造模后大部分时间点均可抑制模型大鼠体温升高，且与模型组比较，P<0.05，具有统计学意义。
表7-1 不同剂量山羊角角蛋白样品对LPS诱导大鼠发热模型的影响
Figure PCTCN2020119336-appb-000025
Figure PCTCN2020119336-appb-000026
与模型组比较：#P<0.05，##P<0.01，###P<0.001；与正常对照组比较:***P<0.001表7-2 不同剂量山羊角角蛋白样品对LPS诱导大鼠发热模型的影响
Figure PCTCN2020119336-appb-000027
与模型组比较：#P<0.05，##P<0.01，###P<0.001；与正常对照组比较:***P<0.001表7-3 不同剂量山羊角角蛋白样品对LPS诱导大鼠发热模型的影响
Figure PCTCN2020119336-appb-000028
与模型组比较：#P<0.05，##P<0.01，###P<0.001；与正常对照组比较:***P<0.001

Claims (16)

  1. 一种山羊角角蛋白的制备方法，其特征在于，包括如下步骤:称量山羊角去塞、粉碎、过筛后并用溶剂提取，山羊角角蛋白提取液经除杂、浓缩、纯化后得山羊角角蛋白，其中所述溶剂选自无机盐的水溶液。
  2. 根据权利要求1的制备方法，其特征在于，包括如下步骤：称量粉碎或切块后的山羊角用溶剂一提取后，取山羊角药渣再用溶剂二提取，得到提取液；提取液经除杂、浓缩、纯化、干燥后得山羊角角蛋白；其中所述的溶剂一选自纯水，溶剂二选自无机盐的水溶液。
  3. 根据权利要求1的制备方法，其中，所述无机盐的水溶液中的无机盐包括尿素、亚硫酸氢钠、十二烷基硫酸钠其中的一种或任选的两种或三种试剂的混合物，所述的无机盐的水溶液中可以任选的加入一定量的蛋白酶抑制剂。
  4. 根据权利要求1的制备方法，其特征在于，所述的方法包括如下步骤：称量山羊角细粉，每千克山羊角细粉使用3～10升的纯水提取2～5次，每次2～8小时，合并提取液；再以每千克冷浸提取后的药渣使用15～100升纯水，尿素10～25千克，亚硫酸氢钠1～5千克，十二烷基硫酸钠0.25～2千克，蛋白酶抑制剂0～0.5升，25～85℃下还原提取3～24h，提取液经离心过滤后，即得山羊角角蛋白提取液。
  5. 根据权利要求4的制备方法，其特征在于，所述的方法包括如下步骤：称量粉碎或切块后的山羊角，以每千克山羊角用3～10升的纯水提取2～5次，每次2～8h后，取药渣再用无机盐的水溶液提取，按每千克药渣使用的无机盐水溶液为5～100升纯水、尿素10～25千克、亚硫酸氢钠1～5千克、十二烷基硫酸钠0.25～2千克、蛋白酶抑制剂0～0.5升计算，25～85℃下还原提取3～24h，提取液经离心过滤后，得到的提取液经除杂、浓缩、纯化、干燥后即得山羊角角蛋白。
  6. 根据权利要求5的制备方法，其中，所述的除杂步骤为：将山羊角角蛋白提取液，使用无机盐粉末除杂得到除杂后的山羊角角蛋白提取液，其中所述的无机盐粉末选自氯化钾。
  7. 根据权利要求6的制备方法，其中，所述的除杂包括如下步骤：将得到的山羊角角蛋白提取液在15～25℃下，加入氯化钾粉末的量与十二烷基硫酸钠的量的比是1～3:1，加氯化钾粉末的过程中产生白色沉淀物，并以此形成混悬液，静置，离心，过滤得到除杂后的山羊角角蛋白提取液。
  8. 根据权利要求5的制备方法，其中所述的浓缩、纯化步骤包括:除杂后的角蛋白提取液一是盐析的方法，二是陶瓷膜超滤系统超滤的方法，三是透析的方法；盐析用无机盐选自硫酸铵、硫酸钠、氯化镁、硫酸镁、硫酸铝、硝酸铵、氯化铵、氯化钠或它们的水溶液，陶瓷膜超滤系统选自陶瓷膜芯除盐系统，透析选用一定截留分子量的透析袋。
  9. 根据权利要求8的制备方法，其特征在于，无机盐盐析浓缩纯化的方法包括如下步骤：除杂后的山羊角角蛋白提取液置于容器中，缓慢加入无机盐或其水溶液，使其盐的终浓度达到10～50％，山羊角角蛋白在无机盐或其水溶液加入的过程中析出，待盐析完全后，静置，过滤，析出的沉淀即为山羊角角蛋白粗提物；山羊角角蛋白粗提物加入纯水混悬，加入纯水的量与权利要求4～5下的纯水提取后山羊角药渣的量的比是10～30:1，混悬后再次用无机盐或其水溶液进行盐析，仍使无机盐的终浓度达到20～50％，待盐析完全后，静置，过滤，弃去清液，得到山羊角角蛋白粗提物，重复上述操作1～3次；盐析后得到的山羊角角蛋白粗提物加入纯水混悬，加入纯水的量与权利要求4～5下的纯水提取后山羊角药渣量的比是10～30:1，混悬搅拌后，静置，过滤，弃去清液，即得山羊角角蛋白粗提物，重复上述操作2～5次后，冷冻干燥，即得山羊角角蛋白。
  10. 根据权利要求8的制备方法，其特征在于，陶瓷膜超滤浓缩纯化的方法包括如下步骤：除杂后的山羊角角蛋白提取液，置于陶瓷膜超滤系统中进行浓缩至山羊角角蛋白浓缩液的体积与陶瓷膜超滤系统的死体积的比是1～3:1，即得山羊角角蛋白浓缩液；山羊角角蛋白浓缩液中加入等体积的纯水稀释后，陶瓷膜超滤系统进行纯化至透过液的体积与加入纯水体积比是1:1时，完成1次纯化操作，重复上述纯化操作5～15次后，用纯水回收陶瓷膜超滤系统中山羊角角蛋白的纯化液， 冷冻干燥，即得山羊角角蛋白。
  11. 根据权利要求8的制备方法，其特征在于，透析纯化的方法包括如下步骤：除杂后的山羊角角蛋白提取液置于透析袋中，并放置于装有纯水的容器中进行纯化，山羊角角蛋白提取液的体积与纯水的体积比是1:20～60，换水间隔时间为6～12h，换水5～10次后，回收透析袋中的角蛋白截留液，冷冻干燥，即得山羊角角蛋白。
  12. 根据权利要求1～11任一项所述制备方法得到的山羊角角蛋白。
  13. 根据权利要求12的山羊角角蛋白，测定蛋白质的种类及含量，其中蛋白质Q0VD04的含量为0.00～20.00％；Q14818的含量为0.00～20.00％；A5PJJ1的含量为0.00～20.00％；F1MXG6的含量为0.00～20.00％；F1MC11的含量为0.00～20.00％；G3X7V4的含量为0.00～20.00％；M0QVY0的含量为0.00～20.00％；Q08D91的含量为0.00～20.00％；A0A140T867的含量为0.00～30.00％；F1N362的含量为0.00～20.00％；E1B898的含量为0.00～50.00％；F1M198的含量为0.00～30.00％；Q5XQN5的含量为0.00～25.00％。
  14. 根据权利要求12的山羊角角蛋白，其特征在于，对得到的山羊角角蛋白，测定其种类及含量，其中蛋白质Q0VD04的含量为0.00～30.00％；Q14818的含量为0.00～20.00％；A5PJJ1的含量为0.00～40.00％；A3KN26的含量为0.00～50.00％；F1MC11的含量为0.00～20.00％；G3X7V4的含量为0.00～20.00％；M0QVY0的含量为0.00～20.00％；Q08D91的含量为0.00～50.00％；F1N362的含量为0.00～20.00％；E1B898的含量为0.00～50.00％；F1M198的含量为0.00～30.00％；Q5XQN5的含量为0.00～20.00％。
  15. 一种药物组合物，其特征在于，包含作为有效成分的权利要求12～14任一项所述的山羊角角蛋白及药学上可接受的载体或赋形剂。
  16. 权利要求12～14任一项所述的山羊角角蛋白和权利要求15所述的药物组合物在制备解热、镇痛、抗惊厥、镇咳、祛痰、降血压、抗炎、抗病毒药物中的应用。
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