WO2021120966A1 - 一种绵羊角角蛋白、其制备方法、其药物组合物及用途 - Google Patents

一种绵羊角角蛋白、其制备方法、其药物组合物及用途 Download PDF

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WO2021120966A1
WO2021120966A1 PCT/CN2020/129730 CN2020129730W WO2021120966A1 WO 2021120966 A1 WO2021120966 A1 WO 2021120966A1 CN 2020129730 W CN2020129730 W CN 2020129730W WO 2021120966 A1 WO2021120966 A1 WO 2021120966A1
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sheep
keratin
content
preparation
group
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PCT/CN2020/129730
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French (fr)
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庾石山
王晓良
屈晶
符江
史国茹
王玲
王晓婧
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
    • A61K35/36—Skin; Hair; Nails; Sebaceous glands; Cerumen; Epidermis; Epithelial cells; Keratinocytes; Langerhans cells; Ectodermal cells
    • 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
    • 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
    • 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
    • C07K1/12—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by hydrolysis, i.e. solvolysis in general
    • 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
    • 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 present invention relates to a sheep 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, antitussive, expectorant, Application in lowering blood pressure, anti-inflammatory and antiviral drugs.
  • Sheep horns are the horns of Ovis (Ovis aries Linnaeus) sheep of the Bovidae family (Bovidae). Functions and indications are described in the "National Chinese Herbal Medicine Compilation" as nourishing and strengthening, clearing away heat and improving eyesight. Through the comparison of literature and historical data, it is found that the function of sheep horn is different from that of endangered animal medicine antelope horn, and there is also a certain difference from goat horn, which is clearly recorded and can replace antelope horn in traditional Chinese medicine compound preparations.
  • goat horn The function of goat horn is described in the "New Compilation of Materia Medica” as specializing in living blood, grinding goat horns for one point, and sending it in wine; in “Medical Lin Zhuan Yao", it is described as functioning near antelope horn; and in “China The antelope horns in Dendrobium Yeguang Pills and Jinzhen Oral Liquid have been replaced by goat horns in the preparations of traditional Chinese medicine prescriptions in the Pharmacopoeia, indicating the similarity of the functions of goat horns and antelope horns.
  • sheep horns also have part of the pharmacological effects embodied by antelope horns, which laid the foundation for the replacement of antelope horns by sheep horns and the enrichment of alternative horn species. Therefore, the research on sheep horns, especially the keratin components that play a major role, is worthy of in-depth exploration and development to ensure the safety, convenience, resource sustainability and functional versatility of sheep horns in clinical applications. On this basis, we extracted and purified sheep keratin to prepare sheep keratin, laying the foundation for subsequent development and utilization.
  • the technical problem solved by the present invention is to provide a type of sheep keratin, its preparation method, pharmaceutical composition and application.
  • the present invention provides the following technical solutions:
  • the first aspect of the technical scheme of the present invention is to provide a method for preparing sheep keratin, including:
  • the medicine residue is extracted with solvent two to obtain the extract; the extract is purified and dried to obtain sheep keratin; wherein the solvent one is selected from Pure water; two solvents are selected from aqueous solutions of inorganic salts;.
  • the preferred raw medicinal material for sheep horn is sheep horn that has been deplugged, washed, and crushed or cut into pieces. The most preferred sheep horns are deplugged, washed, dried and crushed through a 100-mesh sieve to increase the contact area with the solvent and improve the extraction efficiency.
  • the inorganic salt in the aqueous solution of the inorganic salt includes one of urea, sodium bisulfite, sodium lauryl sulfate or an optional mixture of two or three reagents or a certain amount of protease inhibitor added thereto.
  • smashed or cut sheep horns use 3-10 liters of pure water per kilogram of sheep horns, preferably 5-8 liters, more preferably 5-6 liters; extract 2-5 times, preferably It is 3 times; each time is 2-8h, preferably 3-5h; extraction can be carried out under static or dynamic conditions, preferably under dynamic conditions, such as stirring or ultrasound.
  • the sheep horn medicine residue extracted with pure water is extracted with an aqueous solution of inorganic salt.
  • the aqueous inorganic salt solution used per kilogram of medicine residue is 5-100 liters of pure water, preferably 30-70 liters, and more preferably 40 to 50 liters; 10 to 25 kilograms of urea, preferably 8 to 20 kilograms, more preferably 12 to 15 kilograms; sodium bisulfite 1 to 5 kilograms, preferably 2 to 4 kilograms, more preferably 2 to 3 kilograms ; Sodium lauryl sulfate 0.25 to 2 kg, preferably 0.5 to 1.5 kg, more preferably 0.75 to 1 kg; protease inhibitor 0 to 0.5 liters, preferably 0 to 0.1 liters, calculated at 25 to 85 °C, preferably It is 50 ⁇ 80°C, more preferably 65 ⁇ 75°C; reductive extraction is 3 ⁇ 24h, after centrifugal filtration of the extract, the obtained extract is purified and dried to obtain sheep keratin; extraction can be
  • the extract obtained in the above steps needs to be purified by dialysis, membrane ultrafiltration or salting out.
  • the dialysis method the extract is placed in a dialysis bag, the molecular weight cut-off of the dialysis bag is 3500-14000 Da, preferably 3500 -5000Da; place the dialysis bag filled with the extraction solution in a container filled with pure water, the volume ratio of the extraction solution to the volume of pure water is 1:20-60, preferably 1:40-50; water change interval time It is 6-12h, preferably 8-10h; the water is changed 5-10 times, preferably 6-8 times.
  • the retentate in the dialysis bag is recovered and dried to obtain sheep keratin.
  • the type and content of protein are determined, wherein the content of protein Q0VD04 is 5.00-15.00%; the content of Q14818 is 2.00-25.00%; the content of A5PJJ1 is 1.00-15.00%;
  • the content of F1MXG6 is 10.00 ⁇ 30.00%; the content of F1MC11 is 1.00 ⁇ 10.00%; the content of G3X7V4 is 0.10 ⁇ 5.00%; the content of M0QVY0 is 1.00 ⁇ 10.00%; the content of Q08D91 is 1.00 ⁇ 10.00%; the content of A0A140T867 is 0.00 ⁇ 5.00%; the content of F1N362 is 1.00 ⁇ 10.00%; the content of E1B898 is 1.00 ⁇ 15.00%; the content of F1M198 is 1.00 ⁇ 10.00%; the content of Q5XQN5 is 1.00 ⁇ 15.00%.
  • the second aspect of the technical solution of the present invention provides sheep keratin prepared by the preparation method described in the first aspect.
  • the third aspect of the technical solution of the present invention is to provide a pharmaceutical composition, which comprises the sheep keratin of the second aspect as an effective 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 sheep keratin of the present invention can be combined with one or more solid or liquid pharmaceutical excipients and/or adjuvants to prepare a suitable administration that can be used as a human or veterinary medicine.
  • Form or dosage form can be used as a human or veterinary medicine.
  • the sheep keratin of the present invention or the pharmaceutical composition containing it 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 sheep keratin of the present invention can be made 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, esters of higher alcohols, gelatin, semi-synthetic glycerides and the like.
  • sheep keratin or a pharmaceutical composition containing sheep 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 effective ingredient of the sheep 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 sheep 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 sheep keratin or the pharmaceutical composition of the present invention can be administered by any known administration method.
  • the dosage of the sheep 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, and the 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 sheep keratin composition of the present invention, appropriate adjustments can be made to achieve the requirement of the therapeutically effective amount and accomplish the preventive or therapeutic purpose of the present invention.
  • the appropriate daily dosage range of sheep 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 sheep 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 solution of the present invention is to provide the sheep keratin of the second aspect or the pharmaceutical composition of the third aspect for preparing antipyretic, analgesic, anticonvulsant, antitussive, expectorant, lowering blood pressure, and antipyretic Inflammation, the application of antiviral drugs.
  • the medicinal materials are easily available, and the extraction process is simple, which is suitable for large-scale industrial production.
  • Figure 1 Flow chart of the preparation of sheep keratin.
  • the dregs after pure water extraction Add 30ml of pure water, 9g of urea, 2g of sodium bisulfite, 0.45g of sodium lauryl sulfate to 2g, ultrasonic extraction at 75°C for 3h, centrifugation, and then the sheep keratin extract is obtained.
  • the extract is placed in a molecular weight cutoff of In a 3500Da dialysis bag, the ratio of extract to water during dialysis is 1:50.
  • the water is changed every 10h. After 8 water changes, the sheep keratin retentate in the dialysis bag is recovered and freeze-dried to obtain Sheep keratin: The type and content of protein were determined for the obtained sheep keratin.
  • the content of protein Q0VD04 was 11.93%; the content of Q14818 was 19.36%; the content of A5PJJ1 was 2.64%; the content of F1MXG6 was 22.75%;
  • the content of F1MC11 is 2.02%; the content of G3X7V4 is 0.42%; the content of M0QVY0 is 4.55%; the content of Q08D91 is 2.22%; the content of F1N362 is 1.82%; the content of E1B898 is 5.60%; the content of F1M198 is 2.50%; Q5XQN5
  • the content is 5.01%. See Figure 1 for the flow chart of extraction and purification.
  • the ratio of extract to water during dialysis is 1:50.
  • the water is changed every 10h. After 8 times of water changes, the sheep keratin retentate in the dialysis bag is recovered. , Freeze-dried to obtain sheep keratin; determine the type and content of protein of the obtained sheep keratin, the content of protein Q0VD04 is 11.80%; the content of Q14818 is 4.53%; the content of A5PJJ1 is 7.57%; the content of F1MXG6 The content is 23.16%; the content of F1MC11 is 3.71%; the content of G3X7V4 is 0.44%; the content of M0QVY0 is 3.41%; the content of Q08D91 is 3.74%; the content of A0A140T867 is 0.23%; the content of F1N362 is 1.86%; the content of E1B898 It is 9.45%; the content of F1M198 is 4.16%; the content of Q5XQN5 is 7.55%.
  • Drugs and reagents lipopolysaccharide, aspirin, different batches of sheep keratin (S-1: Example 1 of the preparation method of sheep keratin; S-2: Example 2 of the preparation method of sheep 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; sheep 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.
  • Sheep keratin samples S-1 10mg/kg, S-1 50mg/kg can effectively reduce the body temperature of model rats 2h after modeling, and S-2 50mg/kg group can be significantly reduced 2h and 4h after modeling Reduce the body temperature of the model rats, and the continuous cooling effect is longer.
  • P ⁇ 0.05 there is a statistical difference.
  • Example 2 Study on the efficacy of sheep keratin samples on the yeast-induced fever model in SD rats
  • Experimental method Preparation of experimental animals: After the experimental animals have been 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, 12 hours before the experiment, fasting and 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 a 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 divide them into groups of 8 rats.
  • After oral administration of aspirin and different doses of sheep keratin samples immediately subcutaneously injected 20% yeast suspension (10ml/kg), and the normal control group was injected subcutaneously with an equal volume of normal saline. After 2 hours, the rats' body temperature was monitored and 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
  • sheep 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 established successfully and was 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.
  • Sheep keratin samples can inhibit the increase in body temperature of model rats to varying degrees after modeling, and the S-1 50mg/kg group can effectively inhibit the increase in body temperature of model rats at 6h and 8h after modeling.
  • S -2 10mg/kg group and S-2 50mg/kg group can effectively inhibit the increase in body temperature of model rats 4h after modeling, and compared with the model group, P ⁇ 0.05, which is statistically significant.
  • Example 3 Study on the effect of sheep keratin on acetic acid writhing in ICR mice
  • Drugs and reagents aspirin, physiological saline, glacial acetic acid, different batches of sheep keratin (S-1: Example 1 of the preparation method of sheep keratin; S-2: Example 2 of the preparation method of sheep keratin; )
  • mice After 1 day of acclimatization to the environment, the experimental animals were orally given aspirin 300mg/kg, sheep 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 animals within 15 minutes.
  • Experimental groups model group; aspirin: 300mg/kg group; sheep 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, and has a certain analgesic effect. Compared with the model group, P ⁇ 0.05, which is statistically significant.
  • the S-2 50mg/kg group and the S-2 200mg/kg group showed a certain effect on reducing the number of writhing times in mice, and compared with the model group, P ⁇ 0.05, which was statistically significant.
  • Model group 14 24.0 ⁇ 0.2 232.5 ⁇ 19.3 21.6 ⁇ 3.1 Aspirin 300mg/kg 14 23.7 ⁇ 0.2 361.1 ⁇ 36.1** 8.0 ⁇ 1.4*** S-1 50mg/kg 12 23.8 ⁇ 0.2 236.3 ⁇ 18.0 13.7 ⁇ 2.5 S-1 200mg/kg 12 23.7 ⁇ 0.2 210.5 ⁇ 17.7 19.7 ⁇ 2.9
  • Model group 18 24.7 ⁇ 0.3 199.4 ⁇ 12.2 29.8 ⁇ 3.5 Aspirin 300mg/kg 14 24.6 ⁇ 0.3 267.9 ⁇ 17.2** 13.9 ⁇ 2.1** S-2 50mg/kg 14 24.8 ⁇ 0.3 209.1 ⁇ 7.1 20.0 ⁇ 2.1* S-2 200mg/kg 14 24.7 ⁇ 0.3 215.6 ⁇ 46.9 24.3 ⁇ 3.3
  • Example 4 Study on the effect of sheep keratin sample on pain threshold of ICR mice in hot plate experiment
  • Drugs and reagents gabapentin, different batches of sheep keratin (S-1: Example 1 of the preparation method of sheep keratin; S-2: Example 2 of the preparation method of sheep keratin;)
  • mice are screened for pain threshold (hot plate temperature is set at 55 °C ⁇ 0.2 °C), and the pain threshold between 10S-30S is selected as qualified mice, and randomly divided into groups.
  • Group 10 animals weighed and labeled with picric acid.
  • the positive tool drug gabapentin (300mg/kg, 10ml/kg) and sheep keratin samples (50mg/kg, 200mg/kg, 10ml/kg) were given orally.
  • Gabapentin and sheep keratin samples were orally administered again at 9:00 on the second day after weighing (the same dose as the first day), and pain threshold measurement was started 1h later. Three time points were monitored at 1h, 2h, and 3h after administration.
  • mice model group; gabapentin: 300mg/kg group; sheep keratin samples: 50mg/kg group, 200mg/kg group.
  • the Gabapentin 300mg/kg group can increase the pain threshold of ICR mice to varying degrees at 1h, 2h and 3h after administration. Compared with the model group, P ⁇ 0.05, which is statistically significant.
  • the S-1 50mg/kg group can significantly increase the pain threshold of ICR mice 3 hours after administration, and compared with the model group, P ⁇ 0.05, which is statistically significant.
  • Example 5 Study on the effect of sheep keratin samples on pentylenetetrazol (PTZ)-induced epilepsy in mice
  • mice model group; retigabine: 70mg/kg group; retigabine: 60mg/kg group; sheep keratin samples: 50mg/kg group, 200mg/kg group.
  • S-1 200mg/kg group, S-2 50mg/kg group, S-2 200mg/kg group can effectively inhibit the epilepsy grade III seizure rate, and compared with the model group, P ⁇ 0.05 .
  • the S-2 200mg/kg group can effectively inhibit the rate of grade IV epilepsy, and compared with the model group, P ⁇ 0.05, which is statistically different.
  • Group N Number of Grade IV cases Grade IV seizure rate Number of deaths mortality rate
  • Model group 10 10 100% 0 0 Retigabine 60mg/kg 10 6 60% 0 0 S-2 50mg/kg 10 10 100% 1 10% S-2 200mg/kg 10 7 70% 0 0
  • Example 6 Study on the effect of sheep keratin sample on the expectorant effect of phenol red excretion method in mice
  • mice were fasted and watered 16h before the experiment.
  • Mucosultan and different doses of sheep keratin drugs were given orally according to groups (administration volume 10ml/kg), the solvent control group was given the same volume of distilled water, and 2.5% phenol red solution was intraperitoneally injected 1 hour later. 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. The excretion of phenol red was calculated according to the standard curve of phenol red.
  • Example 7 Study on the antitussive effect of sheep keratin samples on mice cough induced by ammonia water
  • Drugs and reagents dextromethorphan hydrobromide, ammonia, 0.2% CMC-Na, different batches of sheep keratin (S-1: Example 1 of the preparation method of sheep keratin; S-2: sheep keratin Example 2 of the preparation method;)
  • the incubation period of cough refers to the number of seconds from the start of the atomization of ammonia to the occurrence of cough. Coughing of mice is based on contraction of their abdominal muscles (breast contraction) and opening their mouths 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.

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Abstract

本发明涉及绵羊角角蛋白，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。本发明通过将绵羊角去塞、干燥、粉碎、提取和纯化，提取出具有解热、镇痛、抗惊厥、镇咳、祛痰、降血压、抗炎、抗病毒活性作用的角蛋白。其药理实验表明本发明的绵羊角角蛋白在体内具有较好的解热、镇痛、抗惊厥、镇咳、祛痰、降血压、抗炎、抗病毒活性。

Description

一种绵羊角角蛋白、其制备方法、其药物组合物及用途 技术领域
本发明涉及一种绵羊角角蛋白，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
绵羊角为牛科(Bovidae)动物绵羊属(Ovis)绵羊(Ovis aries Linnaeus)的角。功能主治在《全国中草药汇编》中描述为滋补强壮，清热明目。通过文献史料比较发现，绵羊角的功能主治与濒危动物药羚羊角存在一定的区别，与有明确记载及中药复方制剂中能够替代羚羊角的山羊角也存在一定的差别。在《中国药典》中明确记载，羚羊角的功能主治为平肝息风，清肝明目，散血解毒。用于肝风内动，惊痫抽搐，妊娠子痫，高热痉厥，癫痫发狂，头痛眩晕，目赤翳障，温毒发斑，痈肿疮毒。而山羊角功能主治在《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；且在《中国药典》中药成方制剂中已将石斛夜光丸、金振口服液等品种中的羚羊角替代为山羊角，表明了山羊角与羚羊角功能主治的相似性。但是，通过现代研究，发现绵羊角同样具有羚羊角所体现的部分药理作用，以此为绵羊角替代羚羊角，丰富替代角类品种奠定了基础。因此对于绵羊角的研究，尤其是对于起主要作用的角蛋白类成分的研究值得深入探索和开发，以保证绵羊角在临床应用中的安全性、方便性、资源可持续性及功能多用性。在此基础上，我们对绵羊角角蛋白进行提取纯化，制备出绵羊角角蛋白，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绵羊角角蛋白、其制备方法，药物组合物和应用。
为解决本发明的技术问题，本发明提供了如下的技术方案:
本发明技术方案的第一方面是提供了一种绵羊角角蛋白的制备方法，包括:
包括如下步骤：称量粉碎或切块后的绵羊角用纯水提取后，取药渣再用溶剂提取，得到提取液；提取液经纯化、干燥后得绵羊角角蛋白；其中所述的溶剂选自无机盐的水溶液。
优选的包括如下步骤：
称量粉碎或切块后的绵羊角用溶剂一提取后，取药渣再用溶剂二提取，得到提取液；提取液经纯化、干燥后得绵羊角角蛋白；其中所述的溶剂一选自纯水；溶剂二选自无机盐的水溶液；。优选的绵羊角原药材是去塞、洗净后粉碎或切块的绵羊角。最优选的绵羊角经去塞、洗净、干燥后并粉碎过100目筛，以增大与溶剂的接触面积，提高提取效率。优选的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或其中加入一定量的蛋白酶抑制剂。
进一步的，称量粉碎或切块后的绵羊角，以每千克绵羊角用3～10升的纯水，优选为5～8升，更优选为5～6升；提取2～5次，优选为3次；每次2～8h，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，例如搅拌或超声。
进一步的，取用纯水提取过的绵羊角药渣，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，优选为30～70升，更优选为40～50升；尿素10～25千克，优选为8～20千克，更优选为12～15千克；亚硫酸氢钠1～5千克，优选为2～4千克，更优选为2～3千克；十二烷基硫酸钠0.25～2千克，优选为0.5～1.5千克，更优选为0.75～1千克；蛋白酶抑制剂0～0.5升，优选为0～0.1升计算，25～85℃下，优选为50～80℃，更优选为65～75℃；还原提取3～24h，提取液经离心过滤后，得到的提取液经纯化、干燥后即得绵羊角角蛋白；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。
进一步的，上述步骤得到的提取液需经过透析、膜超滤或盐析的纯化方法，其中透析方法中，将提取液置于透析袋中，透析袋的截留分子量为3500-14000Da，优选为3500-5000Da；将装有提取液的透析袋放置于装有纯水的容器中，提取液的体积与纯水的体积比是1:20～60，优选为1:40～50；换水间隔时间为6～12h，优选为8～10h；换水5～10次，优选为6～8次，透析完成后回收透析袋中的截留液，干燥，即得绵羊角角蛋白。
进一步的，对上述步骤得到的绵羊角角蛋白，测定其蛋白质的种类及含量，其中蛋白质Q0VD04的含量为5.00～15.00％；Q14818的含量为2.00～25.00％；A5PJJ1的含量为1.00～15.00％；F1MXG6的含量为10.00～30.00％；F1MC11的含量为1.00～10.00％；G3X7V4的含量为0.10～5.00％；M0QVY0的含量为1.00～10.00％；Q08D91的含量为1.00～10.00％；A0A140T867的含量为0.00～5.00％；F1N362的含量为1.00～10.00％；E1B898的含量为1.00～15.00％；F1M198的含量为1.00～10.00％；Q5XQN5的含量为1.00～15.00％。
本发明技术方案的第二方面是提供了第一方面所述的制备方法制备得到的绵羊角角蛋白。
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绵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，可将本发明绵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。
本发明绵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。
本发明绵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，将绵羊角角蛋白或含有绵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绵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，将本发明绵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，本发明的绵羊角角蛋白或药物组合物可用任何公知的给药方法给药。
本发明绵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。可以根据本发明绵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。本发明绵羊角角蛋白的每天的合适剂量范围:本发明的绵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。本发明的绵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了第二方面所述绵羊角角蛋白或第三方面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。
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：绵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：绵羊角角蛋白的制备方法
称量去塞、清洗、晾干后的绵羊角，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，纯水提取后的药渣2g中加入纯水30ml，尿素9g，亚硫酸氢钠2g，十二烷基硫酸钠0.45g，75℃下超声提取3h，离心，即得绵羊角角蛋白提取液，提取液置于截留分子量为3500Da的透析袋中，透析过程中提取液与水的比为1:50，每隔10h换水一次，换水8次后，回收透析袋中的绵羊角角蛋白截留液，冷冻干燥，即得绵羊角角蛋白；对所得的绵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11.93％；Q14818的含量为19.36％；A5PJJ1的含量为2.64％；F1MXG6的含量为22.75％％；F1MC11的含量为2.02％；G3X7V4的含量为0.42％；M0QVY0的含量 为4.55％；Q08D91的含量为2.22％；F1N362的含量为1.82％；E1B898的含量为5.60％；F1M198的含量为2.50％；Q5XQN5的含量为5.01％。提取纯化的流程图见附图1。
实施例2：绵羊角角蛋白的制备方法
称量去塞、清洗、晾干后的绵羊角，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，纯水提取后的药渣2g中加入纯水30ml，尿素9g，亚硫酸氢钠2g，十二烷基硫酸钠0.45g，蛋白酶抑制剂0.3ml，75℃下超声提取3h，离心，即得绵羊角角蛋白提取液，提取液置于截留分子量为3500Da的透析袋中，透析过程中提取液与水的比为1:50，每隔10h换水一次，换水8次后，回收透析袋中的绵羊角角蛋白截留液，冷冻干燥，即得绵羊角角蛋白；对所得的绵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11.80％；Q14818的含量为4.53％；A5PJJ1的含量为7.57％；F1MXG6的含量为23.16％；F1MC11的含量为3.71％；G3X7V4的含量为0.44％；M0QVY0的含量为3.41％；Q08D91的含量为3.74％；A0A140T867的含量为0.23％；F1N362的含量为1.86％；E1B898的含量为9.45％；F1M198的含量为4.16％；Q5XQN5的含量为7.55％。提取纯化的流程图见附图1。
药理实验
实施例1：绵羊角角蛋白对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、不同批次绵羊角角蛋白(S-1：绵羊角角蛋白的制备方法实施例1；S-2：绵羊角角蛋白的制备方法实施例2；)
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绵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；绵羊角角蛋白样品：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、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、绵羊角角蛋白样品S-1 10mg/kg，S-1 50mg/kg在造模后2h均可以有效降低模型大鼠体温，S-2 50mg/kg组在造模后2h及4h可明显降低模型大鼠体温，持续降温作用时间较长，与模型组比较，P<0.05，有统计学差异。
表1-1不同剂量绵羊角角蛋白样品对LPS诱导大鼠发热模型的影响
Figure PCTCN2020129730-appb-000001
Figure PCTCN2020129730-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1-2不同剂量绵羊角角蛋白样品对LPS诱导大鼠发热模型的影响
Figure PCTCN2020129730-appb-000003
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：绵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，不同批次绵羊角角蛋白(S-1：绵羊角角蛋白的制备方法实施例1；S-2：绵羊角角蛋白的制备方法实施例2；)
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、不同剂量绵羊角角蛋 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；绵羊角角蛋白样品：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2-1、2-2)：
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、绵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-1 50mg/kg组在造模后6h及8h均可有效抑制模型大鼠体温升高，S-2 10mg/kg组，S-2 50mg/kg组，在造模后4h均可有效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2-1不同剂量绵羊角角蛋白样品对酵母菌诱导大鼠发热模型的影响
Figure PCTCN2020129730-appb-000004
Figure PCTCN2020129730-appb-000005
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2-2不同剂量绵羊角角蛋白样品对酵母菌诱导大鼠发热模型的影响
Figure PCTCN2020129730-appb-000006
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3：绵羊角角蛋白对对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、不同批次绵羊角角蛋白(S-1：绵羊角角蛋白的制备方法实施例1；S-2：绵羊角角蛋白的制备方法实施例2；)
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、绵羊角角蛋白样品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；绵羊角角蛋白样品：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3-1、3-2)：
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、绵羊角角蛋白样品中S-2 50mg/kg组，S-2 200mg/kg组表现出一定的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。
表3-1不同剂量绵羊角角蛋白样品对ICR小鼠醋酸扭体实验的影响
组别 N 体重(克) 扭体潜伏期(秒) 扭体次数(次)
模型组 14 24.0±0.2 232.5±19.3 21.6±3.1
阿司匹林300mg/kg 14 23.7±0.2 361.1±36.1** 8.0±1.4***
S-1 50mg/kg 12 23.8±0.2 236.3±18.0 13.7±2.5
S-1 200mg/kg 12 23.7±0.2 210.5±17.7 19.7±2.9
与模型组比较：*P<0.05，**P<0.01，***P<0.001
表3-2不同剂量绵羊角角蛋白样品对ICR小鼠醋酸扭体实验的影响
组别 N 体重(克) 扭体潜伏期(秒) 扭体次数(次)
模型组 18 24.7±0.3 199.4±12.2 29.8±3.5
阿司匹林300mg/kg 14 24.6±0.3 267.9±17.2** 13.9±2.1**
S-2 50mg/kg 14 24.8±0.3 209.1±7.1 20.0±2.1*
S-2 200mg/kg 14 24.7±0.3 215.6±46.9 24.3±3.3
与模型组比较：*P<0.05，**P<0.01，***P<0.001
实施例4：绵羊角角蛋白样品对对热板实验中ICR小鼠疼痛阈值的药效研究
动物：ICR 小鼠 雌性
药品与试剂：加巴喷丁、不同批次绵羊角角蛋白(S-1：绵羊角角蛋白的制备方法实施例1；S-2：绵羊角角蛋白的制备方法实施例2；)
实验方法：动物适应实验环境1天后，将ICR小鼠进行痛阈值筛 选(热板温度设定55℃±0.2℃)，选择痛阈值在10S-30S之间的作为合格小鼠，随机分组，每组10只，称重并做苦味酸标记。15:00口服给予阳性工具药加巴喷丁(300mg/kg，10ml/kg)、绵羊角角蛋白样品(50mg/kg、200mg/kg，10ml/kg)。第二天9:00称重后再次口服给予加巴喷丁及绵羊角角蛋白样品(剂量同第一天)，1h后开始进行痛阈值测量，共监测给药后1h、2h、3h三个时间点。
实验分组：模型组；加巴喷丁：300mg/kg组；绵羊角角蛋白样品：50mg/kg组、200mg/kg组。
数据统计：将所得每组数据计算各组小鼠痛阈值均值、标准差及标准误，应用TTEST将各组痛阈值与正常组痛阈值进行组间比较，P<0.05认为有显著性差异。
实验结果(详见表4-1、4-2)：
1、加巴喷丁300mg/kg组给药后1h、2h及3h均能不同程度增高ICR小鼠痛阈值，与模型组比较，P<0.05，有统计学意义。
2、绵羊角角蛋白样品中S-1 50mg/kg组在给药后3h均可明显增高ICR小鼠痛阈值，且与模型组比较，P<0.05，有统计学意义。
表4-1不同剂量绵羊角角蛋白样品对ICR小鼠疼痛阈值的影响
Figure PCTCN2020129730-appb-000007
与模型组比较：*P<0.05，**P<0.01，***P<0.001
表4-2不同剂量绵羊角角蛋白样品对对ICR小鼠疼痛阈值的影响
Figure PCTCN2020129730-appb-000008
Figure PCTCN2020129730-appb-000009
与模型组比较：*P<0.05，**P<0.01，***P<0.001
实施例5：绵羊角角蛋白样品对对戊四唑(PTZ)致小鼠癫痫的药效研究
动物：ICR 小鼠 雄性
药品与试剂：瑞替加滨、不同批次绵羊角角蛋白(S-1：绵羊角角蛋白的制备方法实施例1；S-2：绵羊角角蛋白的制备方法实施例2；)
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：70mg/kg组；瑞替加滨：60mg/kg组；绵羊角角蛋白样品: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5-1、5-2；表6-1、6-2)：
1、在绵羊角角蛋白S-1样品对PTZ致小鼠癫痫实验中，模型组Ⅳ级发作率为90％。80只小鼠有2只死亡。阳性药可以显著延长小鼠Ⅲ级癫 痫发作潜伏期，并且90％抑制癫痫Ⅳ级发作。在绵羊角角蛋白S-2样品对PTZ致小鼠癫痫实验中，模型组Ⅳ级发作率为100％。110只小鼠有5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Ⅲ级潜伏期比较中，S-1 200mg/kg组，S-2 50mg/kg组，S-2 200mg/kg组能有效抑制癫痫Ⅲ级发作率，且与模型组比较P<0.05，具有统计学差异。在癫痫Ⅳ级发作潜伏期比较中，S-2 200mg/kg组能有效抑制癫痫Ⅳ级发作率，且与模型组比较P<0.05，具有统计学差异。
表5-1绵羊角角蛋白样品对PTZ致小鼠癫痫实验-例数统计
组别 N Ⅳ级例数 Ⅳ级发作率 死亡例数 死亡率
模型组 10 9 90％ 0 0
瑞替加滨70mg/kg 10 1** 10％** 0 0
S-1-50mg/kg 10 7 70％ 0 0
S-1-200mg/kg 10 9 90％ 0 0
与模型组比较：*P<0.05，**P<0.01
表5-2绵羊角角蛋白样品对PTZ致小鼠癫痫实验-例数统计
组别 N Ⅳ级例数 Ⅳ级发作率 死亡例数 死亡率
模型组 10 10 100％ 0 0
瑞替加滨60mg/kg 10 6 60％ 0 0
S-2 50mg/kg 10 10 100％ 1 10％
S-2 200mg/kg 10 7 70％ 0 0
与模型组比较：*P<0.05，**P<0.01
表6-1绵羊角角蛋白样品对PTZ致小鼠癫痫实验-Ⅲ级及Ⅳ级癫痫发作潜伏期(mean±SEM)
组别 Ⅲ级发作潜伏期(s) Ⅳ级发作潜伏期(s)
模型组 67±4 180±86
瑞替加滨70mg/kg 93±6** 825±79**
S-1 50mg/kg 72±7 341±129
S-1 200mg/kg 78±3* 195±84
与模型组比较：*P<0.05，**P<0.01
表6-2绵羊角角蛋白样品对PTZ致小鼠癫痫实验-Ⅲ级及Ⅳ级癫痫发作潜伏期(mean±SEM)
组别 Ⅲ级发作潜伏期(s) Ⅳ级发作潜伏期(s)
模型组 58±2 137±27
瑞替加滨60mg/kg 80±8* 446±127*
S-2-50mg/kg 75±7* 127±31
S-2-200mg/kg 81±7** 416±120*
与模型组比较：*P<0.05，**P<0.01
实施例6：绵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、不同批次绵羊角角蛋白(S-1：绵羊角角蛋白的制备方法实施例1；S-2：绵羊角角蛋白的制备方法实施2；)
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绵羊角角蛋白药物(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；绵羊角角蛋白样品：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7-1、7-2)：
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、绵羊角角蛋白样品S-1 50mg/kg组，S-2 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7-1口服绵羊角角蛋白样品对小鼠酚红排泄法祛痰药效实验(X±SEM)
组别 N 酚红排泄量(g/ml) P
溶剂对照组 7 0.521±0.100 ---
沐舒坦30mg/kg 10 0.628±0.081 0.385
S-1 20mg/kg 10 0.744±0.105 0.138
S-1 50mg/kg 10 0.822±0.047** 0.006
与溶剂对照组比较：*P<0.05，与溶剂对照组比较：**P<0.01
表7-2口服绵羊角角蛋白样品对小鼠酚红排泄法祛痰药效实验(X±SEM)
组别 N 酚红排泄量(g/ml) P
溶剂对照组 10 0.497±0.030 ---
沐舒坦30mg/kg 10 0.792±0.089** 0.006
S-2 20mg/kg 10 0.649±0.101 0.168
S-2 50mg/kg 10 0.741±0.074** 0.007
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7：绵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、不同批次绵羊角角蛋白(S-1：绵羊角角蛋白的制备方法实施例1；S-2：绵羊角角蛋白的制备方法实施例2；)
实验方法：按分组口服给予右美沙芬及不同剂量绵羊角角蛋白药物(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；绵羊角角蛋白样品：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8-1、8-2)：
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、绵羊角角蛋白样品S-1 20mg/kg组，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-1 20mg/kg组，S-1 50mg/kg组，S-2 20mg/kg组，S-2 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8-1口服绵羊角角蛋白对小鼠氨水引咳法的镇咳药效实验(X±SEM)
组别 N 体重 潜伏期 P 咳嗽次数 P
溶剂对照组 10 24.4±0.2 22.2±2.4 --- 73.2±4.5 ---
右美沙芬15mg/kg 10 24.2±0.2 33.2±3.0 0.007** 40.3±4.7 0.001**
S-1 20mg/kg 10 23.8±0.2 34.0±3.5 0.009** 47.6±6.8 0.004**
S-1 50mg/kg 10 24.0±0.2 35.2±3.4 0.004** 48.7±5.7 0.002**
与溶剂对照组比较： *P<0.05，与溶剂对照组比较： **P<0.01
表8-2口服绵羊角角蛋白对小鼠氨水引咳法的镇咳药效实验(X±SEM)
组别 N 潜伏期 P 咳嗽次数 P
溶剂对照组 9 24.0±3.2 --- 67.4±3.1 ---
右美沙芬15mg/kg 9 35.1±2.9* 0.020 34.6±4.1** 0.001
S-2 20mg/kg 9 26.7±2.7 0.535 50.6±3.7** 0.003
S-2 50mg/kg 9 34.6±3.3* 0.037 51.1±6.1* 0.029
与溶剂对照组比较： *P<0.05，与溶剂对照组比较： **P<0.01

Claims (11)

  1. 一种绵羊角角蛋白的制备方法，其特征在于，包括如下步骤：称量粉碎或切块后的绵羊角用纯水提取后，取药渣再用溶剂提取，得到提取液；提取液经纯化、干燥后得绵羊角角蛋白；其中所述的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绵羊角，用溶剂一提取后，取绵羊角药渣再用溶剂二提取，得到提取液；提取液经纯化、干燥后得绵羊角角蛋白；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2的制备方法，其特征在于，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。
  4. 根据权利要求2的制备方法，其特征在于，所述的无机盐的水溶液中可以加入一定量的蛋白酶抑制剂。
  5. 根据权利要求2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绵羊角，以每千克绵羊角用3～10升的纯水提取2～5次，每次2～8h提取后，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，得到的提取液经纯化、干燥后即得绵羊角角蛋白。
  6. 根据权利要求2的制备方法，其特征在于，所述制备方法中纯化的方法包括透析、膜超滤、盐析。
  7. 根据权利要求6的制备方法，其特征在于，所述的透析方法包括如下步骤：绵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透析纯化除盐，绵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，回收透析袋中的截留液，干燥，即得绵羊角角蛋白。
  8. 根据权利要求1-7任一项的制备方法，其特征在于，对得到的 绵羊角角蛋白，测定其蛋白质的种类及含量，其中蛋白质Q0VD04的含量为0.00～30.00％；Q14818的含量为0.00～30.00％；A5PJJ1的含量为0.00～30.00％；F1MXG6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0.00～20.00％；A0A140T867的含量为0.00～20.00％；F1N362的含量为0.00～20.00％；E1B898的含量为0.00～20.00％；F1M198的含量为0.00～20.00％；Q5XQN5的含量为0.00～20.00％。
  9. 根据权利要求1～8任一项所述制备方法得到的绵羊角角蛋白，以及权利要求1～8任一项所述制备方法制备得到的绵羊角角蛋白的混合物。
  10. 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9所述的绵羊角角蛋白及药学上可接受的载体或赋形剂。
  11. 权利要求9所述的绵羊角角蛋白或权利要求10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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