WO2006079288A1 - Use of human lysozyme for preparing cosmetics against acne - Google Patents

Use of human lysozyme for preparing cosmetics against acne Download PDF

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Publication number
WO2006079288A1
WO2006079288A1 PCT/CN2006/000155 CN2006000155W WO2006079288A1 WO 2006079288 A1 WO2006079288 A1 WO 2006079288A1 CN 2006000155 W CN2006000155 W CN 2006000155W WO 2006079288 A1 WO2006079288 A1 WO 2006079288A1
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WIPO (PCT)
Prior art keywords
human lysozyme
cosmetic
emulsifier
preparation
treating acne
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PCT/CN2006/000155
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French (fr)
Chinese (zh)
Inventor
Mi An
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Mi An
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Publication of WO2006079288A1 publication Critical patent/WO2006079288A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • 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
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A61K38/47—Hydrolases (3) acting on glycosyl compounds (3.2), e.g. cellulases, lactases
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
    • A61K8/66—Enzymes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/10—Anti-acne agents
    • 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
    • A61P31/02—Local antiseptics

Definitions

  • the present invention relates to a novel use of human lysozyme, in particular a new use of human lysozyme in the field of cosmetics.
  • Penicillin-resistant Streptococcus pneumoniae which used to be sensitive to penicillin, erythromycin, sulfonamides and the like, is now almost invulnerable.
  • Klebsiella pneumoniae has a resistance of 51.85 % to 100% to 16 high-grade antibiotics such as Xi Lixin and Fu Daxin.
  • Methicillin-resistant Staphylococcus aureus (MRSA) has no cure except vancomycin. It can be seen from the history of bacterial resistance development that after a new antibiotic appears, there are a number of resistant strains. Appearance, and the addition of antibiotics to cosmetics is not allowed, so there is currently no ideal cosmetic for acne.
  • the object of the present invention is to overcome the above-mentioned problems and to provide a new use of human lysozyme in the preparation of a cosmetic for treating acne or acne, but it is sensitive, non-resistant, and has no side effects.
  • the present invention relates to the use of human lysozyme in the preparation of a cosmetic for treating acne, in particular the application of human lysozyme in the preparation of a cosmetic for treating acne caused by Staphylococcus aureus, epiphylococcus, Propionibacterium or aphids. .
  • the human lysozyme is genetically engineered recombinant human lysozyme or genetically engineered to express the amino terminus of human lysozyme
  • the cosmetic contains human lysozyme 300 U to 3 million U/mL/g. You can also add the killing and anti-itching agent SM-650. Cosmetics
  • Production process 1. Add the raw material dimethyl silicone oil 200cst, 18 alcohol, octyl carbonic acid glyceride / GTCC, isooctyl palmitate / GD - 5103, vitamin E / VE, emulsifier Montanov 68 into the oil phase chemical pot In the middle, stir and heat to 85 °C; 2. Add the raw material glycerin, emulsifier HR - hyaluronic acid HA, Jieqiong II and deionized water to the emulsification pot and stir to 87-89 ° C ; 3.
  • Production process 1. Add the raw material dimethyl silicone oil 200cst, 18 alcohol, octyl carbonic acid glyceride / GTCC, isooctyl palmitate / GD - 5103, vitamin E / VE, emulsifier Montanov 68 into the oil phase chemical pot Medium, stir and heat to 85
  • Production process 1. Firstly, the raw material Kappa resin 941 and carboxymethyl cellulose CMC are uniformly dispersed in glycerin, and the ionized water is fully mixed and mixed under stirring, and the process should be prepared 4 hours before production. The raw material polyvinyl alcohol 1788 was then added with stirring. 2. Add the evenly stirred 1 into the emulsification pot, then add Tween 80, azone and water-soluble silicone oil and stir to heat to 75 °C, start homogenization, homogenize at high speed for 10 minutes, then stop high-speed homogenization, low speed Quality 15 points After the clock was stopped, stirring was continued for another 10 minutes, and the stirring speed was 60 rpm.
  • lotion the purity of 95% ⁇ 99% of the recombinant human lysozyme to obtain 1500U ⁇ 300,000 U / ml / g, phosphate buffer 10 ⁇ 20mM (pH 6. 0 ⁇ 7. 5) 75 ⁇ 85% 5 ⁇ 25% propylene glycol or glycerol, 5 ⁇ 15% 2-pyrrolidone- 5-carboxylic acid sodium, 1/5 to 10% Tween 80, 1 ⁇ 5% water-soluble azone, paraben 0. 01 ⁇ 0. 05%, potassium sorbate 0. 01 ⁇ 0. 1%, mixed and homogenized at room temperature, made into a lotion.
  • the recombinant human lysozyme in the above formula was provided by Dalian Qilong Biotechnology Research Institute, and other raw materials were provided by Beijing Rihua Research Institute.
  • Recombinant human lysozyme is prepared on the basis of 200 ml medium, using 3 ⁇ 4P0 4 4-8 ml MgS0 4 l -5 g, K 2 S0 4 2_6 g, KOH 1-3 g, CaS0 4 2H 2 01-3.5 g, plus Distilled water to 200 ml, inoculated with glycerol tube seeds after autoclaving, the number of revolutions of the shaker was 250 rpm, the culture temperature was 20-35 ° C, and the culture was carried out for 36 to 48 hours on a constant temperature bed. Production tank culture is carried out after seed tank cultivation.
  • the culture solution in which the fermentation expression was completed was extracted and purified, and the extracted and purified protein concentrate was freeze-dried, and the amount of protein, purity, and recombinant human lysozyme activity (30000 U/mg) were measured and stored.
  • Human recombinant Lysozyme HLZ Active unit 30000 units / mL, provided by Dalian Qilong Biotechnology Research Institute,
  • control lysozyme (Contra Lysozyme, CLZ): white powder, active unit: 50000 units / mg, the United States SIGMA company products, batch number: L6876. 3, clarithromycin: potency 948 / mg, China National Institute for the Control of Pharmaceutical and Biological Products, batch number:
  • Roxithromycin Potency 878 u /mg, China National Institute for the Control of Pharmaceutical and Biological Products, batch number -
  • Amoxicillin for injection Harbin Pharmaceutical Factory, batch number: 010504.
  • strains used were all clinical isolates collected in Sichuan and Beijing in 2002. 4 ⁇ 2003. It was re-identified by the API method and used for testing.
  • Tris-HC1 buffer 0. 1M Tris 100ml, 0. 1M HC1 70ml, High Water 800ml, measure PH value, adjust to pH7.2 with HC1 solution, add High Water to 1000ml.
  • Tris-HCl lipid agar base Add the lipid agarose in Tris-Cl buffer and use it at 116 °C.
  • M-H medium China National Institute for the Control of Pharmaceutical and Biological Products
  • M-H broth culture medium Weigh 25g and 1000ml distilled water, heat to dissolve, dispense, autoclave, 116 20 minutes.
  • M-H solid medium Weigh 36g, add 1000ml of distilled water, autoclave, 116 ⁇ 20 minutes, for the susceptibility test of Gram-positive, negative aerobic bacteria.
  • Blood culture medium which is prepared by adding 5-10% defibrated rabbit blood to M-H medium, for drug sensitivity test of Enterococcus and Streptococcus.
  • the minimum inhibitory concentration (MIC) of the test drug against the test strain was determined by agar double dilution method.
  • the test drug is dissolved in sterile distilled water and diluted appropriately.
  • the lcc solution was mixed with 9 ml of melted Tris-HC1 agarose solid medium, and diluted twice to prepare a series of drug-containing plates.
  • test results (1) The in vitro antibacterial activity of human lysozyme on clinical isolates is shown in Table 1. (2) The MIC50 and MIC90 of human lysozyme against 379 clinical isolates of pathogenic bacteria are shown in Table 1.
  • human lysozyme has a certain antibacterial effect in vitro, and has a good effect on the antibacterial activity of the resistant strain.
  • Human lysozyme is a small molecule protein found in the body's tears, sputum, sputum, white blood cells and serum. It has a bactericidal effect on a variety of Gram-positive bacteria and a few Gram-negative bacteria.
  • Recombinant human lysozyme Human
  • Lysozyme mainly cuts off the linkage between the ⁇ _1 and 4 glycosidic bonds between N-acetylglucosamine and N-acetylmuramic acid in peptidoglycan, destroys the peptidoglycan scaffold and causes bacterial lysis.
  • Propionibacterium 02-7-4 0.008(0.02) 4(20) 0.002 0.002 Propionibacterium 02-7-5 0.008(0.02) 0.25(0.63) ⁇ 0.001 ⁇ 0.001 Propionibacterium 02-7-6 0.008(0.02) 0.063(0.31) 0.008 0.25 Propionibacterium 02-7-9 0.063(0.2) 0.125(0.63) 0.016 Propionibacterium 02-7-7 0.125(0.4) 0.016(0.08) 0.032 0.25 Propionibacterium 03-2-22 0.032 (0.1) 0.5(2.5) 1 1 Propionibacterium 03-2-30 0.063(0.2) 4(10) >1 >1 Propionibacterium 03-2-32 0.016(0.05) 0.25(1.25) 0.008 0.5 Propionibacterium 03-2-33 0.032(0.1) 2(10) >1 >1 Propionibacterium 03-2-36 0.063(0.2) 4(20) 0.25 0.5 Propionibacterium 03-2-37 0.032(
  • Human recombinant Lysozyme HLZ Active unit 300 units / mL, provided by Dalian Qilong Biotechnology Research Institute,
  • Killing and antipruritic agent SM-650 White powder, purity 99%, ph: 6 ⁇ 8, Production unit: Beijing Naite Biochemical Technology Research Institute test aphid: The aphid specimen used is the clinical outpatient hemorrhoid of the Fourth Military Medical University patient. Test method: Determination of in vitro aphid activity: ⁇ The human lysozyme (HLZ) plus the anti-itching agent SM-650 minimum killing concentration (MIC) was determined by photo method.
  • HLZ human lysozyme
  • MIC minimum killing concentration
  • the aphid specimens were separated onto a slide, and the human lysozyme (HLZ) 300 units/mL, the anti-itching agent SM-650 1%/Ml o was dropped onto the slide aphid specimens, and the results were observed to the minimum acaricidal concentration (MIC). ).
  • the test results are shown in Table 2. The results indicate that human lysozyme plus acaricidal antipruritic SM-650 has a killing effect on in vitro killing. Table 2
  • Recombinant human lysozyme concentrate active unit 3000 units / mL test drugs and reagents
  • Human recombinant Lysozyme HLZ Active unit 3000 units / mL, provided by Dalian Qilong Biotechnology Research Institute,
  • Killing and antipruritic agent SM-650 White powder, purity 99%, ph: 6 ⁇ 8, Production unit: Beijing Naite Biochemical Technology Institute' Test aphid: The aphid specimen used is the clinical clinic of the Fourth Military Medical University. Acne patients. Test method: In vitro aphid activity assay: The lowest killing concentration (MIC) of human lysozyme (HLZ) plus acaricidal antipruritic SM-650 was determined by photo method.
  • MIC lowest killing concentration
  • the aphid specimens were separated onto the slides, and the human lysozyme (HLZ) 3000 units/mL, the anti-itching agent SM-650 1%/Ml c was dropped onto the slides of the aphid specimens, and the results were observed to obtain half the acaricidal concentration (MBC). ).
  • the test results are shown in Table 3. The results show that human lysozyme plus acaricidal antipruritic SM-650 has a killing effect on in vitro killing. table 3
  • recombinant human lysozyme concentrate activity unit 30000 units / mL test drugs and reagents
  • Human recombinant Lysozyme HLZ Active unit 30000 units / mL, provided by Dalian Qilong Biotechnology Research Institute,
  • Killing and antipruritic agent SM-650 White powder, purity 99%, ph: 6 ⁇ 8, Production unit: Beijing Naite Biochemical Technology Research Institute test aphid: The aphid specimen used is the clinical outpatient hemorrhoid of the Fourth Military Medical University. patient. Test method: In vitro aphid activity assay: The lowest killing concentration (MIC) of human lysozyme (HLZ) plus acaricidal antipruritic SM-650 was determined by photo method.
  • MIC lowest killing concentration
  • HZ human lysozyme
  • acaricidal antipruritic SM-650 was determined by photo method.
  • the aphid specimens were separated onto a slide, and human lysozyme (HLZ) was used at 30,000 units/mL, and the anti-itching agent SM-650 1%/Ml o was dropped onto the slide aphid specimens. The results were observed, and the live aphids were killed. concentration. The test results are shown in Table 4. The results show that human lysozyme plus acaricidal antipruritic SM-650 has a killing effect on in vitro killing. Table 4
  • the in vitro killing effect of human lysozyme (HLZ) plus acaricidal antipruritic SM-650 on five clinical isolates of aphid samples was determined by photo method. The results showed that: human lysozyme plus acaricidal antipruritic SM-650 pair Aphids have a killing effect.
  • the minimum acaricidal concentration of the killing and antipruritic agent SM-650 is MIC Ri ⁇ 0. 2% ⁇ 2% o human lysozyme (HLZ) activity unit: 300 units / ml ⁇ 300 million units / ml.
  • Human Lysozyme Human Lysozyme developed by Dalian Qilong Biotechnology Research Institute has Other lysozymes have the same mechanism of action, mainly cutting off the e between the N-acetylglucosamine and the N-acetylmuramic acid in the peptidoglycan.
  • Killing and anti-itching agent SM-650 has a strong killing effect. Effects of human lysozyme on the swelling of mouse ear-induced ear edema 50 healthy male Kunming mice weighing 27-30 grams were used, the animals were free to drink water, and the temperature of the laboratory was controlled at 22-28 °C. Animal feeding Rat standard feed. Randomly divided into 5 groups of 10 each. The first group was a blank control group; the second, third and fourth groups were human lysozyme at doses of 120, 60, 30 IU/head; the fifth group was chicken lysozyme (positive control), 30 IU/only.
  • mice were coated with 1% croton oil (croton oil, light brown oily liquid, pharmaceutical grade, batch number 000309, produced by the Huabao Natural Medicine Factory of Jishui County, Jiangxi province). : Ether 25: 5: 70 mixed solvent is prepared to make 1% concentration for use.) 30 ⁇ inflammation, after half an hour, use double distilled water 20 ⁇ 1, human lysozyme (6000IU/ML) 20 ⁇ 1, human lysozyme (3000IU/ML) 20 ⁇ Human lysozyme (1500 IU/ML) 20 ⁇ l, chicken lysozyme (1500 IU/ML) 20 ⁇ l was applied to the inner side of the right ear of each group of mice.
  • croton oil croton oil, light brown oily liquid, pharmaceutical grade, batch number 000309, produced by the Huabao Natural Medicine Factory of Jishui County, Jiangxi province).
  • mice were sacrificed from the cervical vertebrae 4 hours after the inflammation.
  • the left and right ears were cut along the baseline of the auricle.
  • the punches were used to punch the two ears and accurately weighed.
  • the difference between the left and right ear weights was the degree of swelling.
  • the difference between the drug group and the blank control group was compared by the sputum test, and the inhibition rate was determined. The test was repeated once.
  • the test results are shown in Table 17-22.
  • the mice were exposed to human lysozyme 120, 60, 30 IU/only, which significantly reduced the degree of ear swelling caused by croton oil in mice. After the sputum test, there was a significant difference between the drug group and the blank control group ( ⁇ 0 05), indicating that human lysozyme has an anti-inflammatory effect. ⁇
  • the invention has opened up a new application for genetic recombinant human lysozyme and opened up a new application field.
  • the cosmetics prepared by recombinant human lysozyme have safe and non-toxic side effects and have good application prospects.
  • Recombinant human lysozyme is rich in source, simple in preparation process, and can be used as facial cleanser or cleansing lotion, soap, lotion, lotion, cream or mask. It is easy to use and can be used in the same way as various existing cosmetics. Cosmetics are used in sets.
  • Recombinant human lysozyme is prepared by preparing 200 ml of medium, using 6 ml of phosphoric acid, 3 g of magnesium sulfate, 4 g of potassium sulfate, 1 g of potassium hydroxide, 1.5 g of calcium sulfate, and adding distilled water to 200 ml. After autoclaving. The glycerol tube seed was inoculated, the number of revolutions of the shaker was 250 rpm, the culture temperature was 20-35 ° C, and the culture was carried out for 36-48 hours on a constant temperature bed. The seed tank culture is carried out, and finally the production tank culture is carried out.
  • the culture solution in which the fermentation expression is completed is extracted and purified, and the extracted and purified recombinant human lysozyme concentrate is subjected to protein measurement and activity storage.
  • a 95% pure recombinant human lysozyme concentrate was dissolved in water to prepare 30000 U/mL for use.
  • the recombinant human lysozyme having a purity of 95% was prepared to have a 30,000 U/mL/g, a phosphate buffer solution of 10 to 20 mM (pH 6.5 to 7.5), 80%, 15% propylene glycol, and 6% 2-pyrrolidone-5.
  • a phosphate buffer solution of 10 to 20 mM pH 6.5 to 7.5
  • propylene glycol 80%, 15% propylene glycol
  • 2-pyrrolidone-5 6%
  • the human lysozyme was prepared according to the method described in Example 1.
  • the human lysozyme treatment acne skin cream was produced according to the following formula and process:
  • the speed is 60 rpm; 4, slow cooling through water, the temperature thickener SepiGEL305 and the anti-itch agent SM- 650 homogenized after 2 minutes of vacuum degassing at 60 ° C; 5, when 45 ° C
  • a skin cream was prepared as described in Example 2, and the formulation (weight) was as follows:
  • the human lysozyme was prepared according to the method described in Example 1, and prepared into a therapeutic acne cream according to the following formula and process: Formula: (weight)
  • Emulsifier Montanov 68 1.0 Glycerin 8
  • the speed is 60 rpm; 4, slow cooling through water, the temperature thickener SepiGEL305 and the anti-itch agent SM- 650 homogenized after 2 minutes of vacuum degassing at 60 ° C; 5, when 45 ° C
  • the cream was made according to the method described in Example 4, and the specific formula (weight) was as follows:
  • Human lysozyme was prepared as described in Example 1, and then treated to prepare acne emulsion according to the following formulation (weight):
  • the emulsion was prepared as described in Example 6, and the specific formulation (weight) was as follows:
  • Human lysozyme was produced according to the method described in Example 1, and then a mask for treating acne was prepared according to the following formula (weight) -
  • Killing and anti-itching agent SM- 650 is 99% pure, 0.6
  • a film-forming material slurry is prepared by adding a formula drug, a coloring agent, etc., a defoaming film, a drying film, a film, a content, and a finished product package.
  • the specific process is -
  • the raw material Kappa resin 941 and carboxymethyl cellulose CMC are uniformly dispersed in glycerin, and the ionized water is thoroughly mixed and mixed under stirring, and the process should be prepared 4 hours before the production.
  • the raw material polyvinyl alcohol 1788 was then added with stirring.
  • a mask for treating acne was prepared according to the method described in Example 8, and the specific formulation (weight) was as follows:
  • Killing and anti-itching agent SM-650 has a purity of 99% 0.8.

Abstract

A novel use of Human Lysozyme, especially for preparing cosmetics against acne caused by Staphylococcus aureus, Staphylococcus epidermidis, Propionibacterium or Acarophobia. The cosmetics can be produced in the form of lotion, skin-caring cream, milk, facial mask and the like. The use of the gene recombinant Human Lysozyme in cosmetics has provied a new application area and is safe, free of toxic and side effect. In addition, the gene recombinant Human Lysozyme enjoys abundant resources, simple preparation process as well as convenient application.

Description

人溶菌酶在制备治疗痤疮的化妆品中的应用 技术领域：  Application of human lysozyme in the preparation of cosmetics for treating acne
本发明涉及人溶菌酶的新用途， 特别是人溶菌酶在化妆品领域的新用途。  The present invention relates to a novel use of human lysozyme, in particular a new use of human lysozyme in the field of cosmetics.
背景技术： Background technique:
由金黄色葡萄球菌、 表葡菌、 丙酸杆菌引起的痤疮非常普遍， 一般的人都采用化妆品 来减轻症状或治疗， 现有化妆品治疗痤疮产品大部分使用抗生素， 由于抗生素长期使用细 菌产生耐药性， 所以治疗痤疮效果不理想， 并且毒副作用非常大。 抗生素创造了许多医学 奇迹， 使许多疾病消失无踪， 如肺炎、 脑膜炎、 产褥热、 败血症、 结核等。 然而， 耐药菌 的发展令人触目惊心。 如耐青霉素的肺炎链球菌， 过去对青霉素、 红霉素、 磺胺等药品都 很敏感， 现在几乎"刀枪不入"。肺炎克雷伯氏菌对西力欣、 复达欣等 16种高档抗生素的耐 药性高达 51.85 %— 100%。 而耐甲氧西林的金黄色葡萄球菌 （MRSA) 除万古霉素外已经 无药可治， 从细菌的耐药发展史可以看出， 在某种新的抗生素出现以后， 就有一批耐药菌 株出现， 而且在化妆品中添加抗生素药物是不被允许的， 所以目前针对痤疮还没有理想的 化妆品。  Acne caused by Staphylococcus aureus, Epibacterium, Propionibacterium is very common, and people generally use cosmetics to alleviate symptoms or treatment. Most of the existing cosmetics for the treatment of acne products use antibiotics, because of the long-term use of bacteria to produce resistance. Sex, so the treatment of acne is not ideal, and the side effects are very large. Antibiotics have created many medical miracles that have caused many diseases to disappear, such as pneumonia, meningitis, puerperal fever, sepsis, tuberculosis, etc. However, the development of resistant bacteria is shocking. For example, penicillin-resistant Streptococcus pneumoniae, which used to be sensitive to penicillin, erythromycin, sulfonamides and the like, is now almost invulnerable. Klebsiella pneumoniae has a resistance of 51.85 % to 100% to 16 high-grade antibiotics such as Xi Lixin and Fu Daxin. Methicillin-resistant Staphylococcus aureus (MRSA) has no cure except vancomycin. It can be seen from the history of bacterial resistance development that after a new antibiotic appears, there are a number of resistant strains. Appearance, and the addition of antibiotics to cosmetics is not allowed, so there is currently no ideal cosmetic for acne.
发明内容： Summary of the invention:
本发明的目的是克服上述不足问题， 提供一种人溶菌酶在制备治疗粉刺或痤疮的化妆 品中的新用途， 不过敏， 不耐药， 无副作用> 效果佳。  SUMMARY OF THE INVENTION The object of the present invention is to overcome the above-mentioned problems and to provide a new use of human lysozyme in the preparation of a cosmetic for treating acne or acne, but it is sensitive, non-resistant, and has no side effects.
实际上， 本发明涉及人溶菌酶在制备治疗痤疮的化妆品中的应用， 尤其是人溶菌酶在 制备治疗由金黄色葡萄球菌、 表葡菌、 丙酸杆菌或螨虫引起的痤疮的化妆品中的应用。  In fact, the present invention relates to the use of human lysozyme in the preparation of a cosmetic for treating acne, in particular the application of human lysozyme in the preparation of a cosmetic for treating acne caused by Staphylococcus aureus, epiphylococcus, Propionibacterium or aphids. .
所述人溶菌酶为基因工程表达的重组人溶菌酶或基因工程表达人溶菌酶的氨基端带有 The human lysozyme is genetically engineered recombinant human lysozyme or genetically engineered to express the amino terminus of human lysozyme
(谷氨酸一丙氨酸） 2或 （谷氨酸一丙氨酸） 3修饰的人溶菌酶或基因工程表达或化学合成 突变体重组人溶菌酶。 (Glutamate-alanine) 2 or (glutamate-alanine) 3 modified human lysozyme or genetically engineered or chemically synthesized mutant recombinant human lysozyme.
所述化妆品中含有人溶菌酶 300U〜300万 U/mL/g。还可以加入杀螨止痒剂 SM-650。化妆品 The cosmetic contains human lysozyme 300 U to 3 million U/mL/g. You can also add the killing and anti-itching agent SM-650. Cosmetics
1  1
确 认 本 为系统列护肤品， 如洗面奶、 化妆水、 护肤眘、 面霜、 乳液和面膜等， 具体配方及生产工 艺如下所述: Confirmation For the system of skin care products, such as facial cleanser, lotion, skin care, cream, lotion and mask, the specific formula and production process are as follows:
1、 护肤 ¾: 1, skin care 3⁄4 :
配方： （重量份数）  Recipe: (parts by weight)
(1) 重组人溶菌酶 300万 U/mL/g  (1) Recombinant human lysozyme 3 million U/mL/g
(2) 二甲基硅油 200cst 2.0-4.0  (2) Dimethyl silicone oil 200cst 2.0-4.0
(3) 18醇 3.0-6.0  (3) 18 alcohol 3.0-6.0
(4) 辛碳癸酸甘油酯 /GTCC 2-5.0  (4) Cyanuric acid glyceride / GTCC 2-5.0
(5) 棕榈酸异辛酯 /GD— 5103 4.0-7.0  (5) Isooctyl palmitate /GD-5103 4.0-7.0
(6) 维生素 E/VE 0.3-0.7  (6) Vitamin E/VE 0.3-0.7
(7) 乳化剂 Montanov 68 1.0-3.0  (7) Emulsifier Montanov 68 1.0-3.0
(8) 甘油 5-15  (8) Glycerin 5-15
(9) 乳化剂 HR—S! 1.0-3.0  (9) Emulsifier HR-S! 1.0-3.0
(10) 透明脂酸 HA 0.01-0.04  (10) Transparent fatty acid HA 0.01-0.04
(11) 增稠剂 SepiGEL305 0.1-1  (11) Thickener SepiGEL305 0.1-1
(12) 杰玛一 II 1.0-2.0  (12) Gemma II II 1.0-2.0
(13) 香精 0.10-0.20  (13) Fragrance 0.10-0.20
(14)去离子水 71.-77.  (14) Deionized water 71.-77.
(15) 杀螨止痒剂 SM- 650 0.5—1  (15) Killing and anti-itching agent SM- 650 0.5-1
生产工艺： 1、 将原料二甲基硅油 200cst、 18 醇、 辛碳癸酸甘油酯 /GTCC、 棕榈酸异 辛酯 /GD— 5103、 维生素 E/VE、 乳化剂 Montanov 68 加入油相化料锅中， 搅拌加热到 85 °C; 2、 将原料甘油、 乳化剂 HR— 透明脂酸 HA、 杰琼一 II和去离子水加至乳化锅中 加热搅拌至 87-89°C; 3、 启动真空泵将乳化锅抽真空到 0.06Mpa， 停止真空， 将油相抽进 乳化锅内高速均质 10分钟， 搅拌转速 70转， 高速均质后停止； 低速均质 10分钟， 停止均 质， 继续搅拌 10 分钟， 转速为 60 转； 4、 通水慢速冷却， 温度 60Ό时加入原料增稠剂 SepiGEL305和杀螨止痒剂 SM-650均质 2分钟后真空脱气； 5、 当 45°C时加入原料去离子 水和人溶菌酶溶液， 搅拌至 40°C时停机出料,（在符合 GMP要求的制药工厂， 按制药规程 完成） 制成护肤膏。 Production process: 1. Add the raw material dimethyl silicone oil 200cst, 18 alcohol, octyl carbonic acid glyceride / GTCC, isooctyl palmitate / GD - 5103, vitamin E / VE, emulsifier Montanov 68 into the oil phase chemical pot In the middle, stir and heat to 85 °C; 2. Add the raw material glycerin, emulsifier HR - hyaluronic acid HA, Jieqiong II and deionized water to the emulsification pot and stir to 87-89 ° C ; 3. Start the vacuum pump Vacuum the emulsifier to 0.06Mpa, stop the vacuum, pump the oil phase into the emulsifier pot for 10 minutes at high speed, stir at 70 rpm, stop at high speed and homogenize; Quality, continue to stir for 10 minutes, the speed is 60 rpm; 4, slow cooling through water, temperature 60 加入 Add raw material thickener SepiGEL305 and killing itch anti-itch agent SM-650 after 2 minutes of vacuum degassing; 5, when Add the raw material deionized water and human lysozyme solution at 45 °C, stop the output when stirring to 40 °C, (in the pharmaceutical factory that meets GMP requirements, according to the pharmaceutical regulations) to make a skin cream.
2、 面霜：  2, face cream:
配方： （重量份） Recipe: (parts by weight)
(1) 重组人溶菌酶 300万 U/mL/g  (1) Recombinant human lysozyme 3 million U/mL/g
(2) 二甲基硅油 200cst 2.0-4.0  (2) Dimethyl silicone oil 200cst 2.0-4.0
(3) 18醇 3.0-6.0  (3) 18 alcohol 3.0-6.0
(4) 辛碳癸酸甘油酯 /GTCC 2-5.0  (4) Cyanuric acid glyceride / GTCC 2-5.0
(5) 棕榈酸异辛酯 /GD— 5103 4.0-7.0  (5) Isooctyl palmitate /GD-5103 4.0-7.0
(6) 维生素 E/VE 0.2-1.2  (6) Vitamin E/VE 0.2-1.2
(7) 乳化剂 Montanov 68 1.0-3.0  (7) Emulsifier Montanov 68 1.0-3.0
(8) 甘油 5-8  (8) Glycerin 5-8
(9) 乳化剂 HR— Si 1.0-3.0  (9) Emulsifier HR- Si 1.0-3.0
(10) 透明脂酸 HA 0.01-0.04  (10) Transparent fatty acid HA 0.01-0.04
(11) 增稠剂 SepiGEL305 0.1— .5  (11) Thickener SepiGEL305 0.1— .5
(12) 杰琼一 II 1.0-2.0  (12) Jie Qiongyi II 1.0-2.0
(13) 香精 0.1-0.2  (13) Fragrance 0.1-0.2
(14)去离子水 71.0-76.0  (14) Deionized water 71.0-76.0
(15) 杀螨止痒剂 SM- 650 0.5—1  (15) Killing and anti-itching agent SM- 650 0.5-1
生产工艺： 1、 将原料二甲基硅油 200cst、 18 醇、 辛碳癸酸甘油酯 /GTCC、 棕榈酸异 辛酯 /GD— 5103、 维生素 E/VE、 乳化剂 Montanov 68 加入油相化料锅中， 搅拌加热到 85 Production process: 1. Add the raw material dimethyl silicone oil 200cst, 18 alcohol, octyl carbonic acid glyceride / GTCC, isooctyl palmitate / GD - 5103, vitamin E / VE, emulsifier Montanov 68 into the oil phase chemical pot Medium, stir and heat to 85
V 2、 将原料甘油、 乳化剂 HR— S,、 透明脂酸 HA、 杰琼一 II和去离子水加至乳化锅中 加热搅拌至 87-89Ό ; 3、 启动真空泵将乳化锅抽真空到 0. 06Mpa， 停止真空， 将油相抽进 乳化锅内高速均质 10分钟， 搅拌转速 70转， 高速均质后停止； 低速均质 10分钟， 停止均 质， 继续搅拌 10 分钟， 转速为 60 转； 4、 通水慢速冷却， 温度 60Ό时加入原料增稠剂 SepiGEL305和杀螨止痒剂 SM- 650均质 2分钟后真空脱气； 5、 当 45°C时加入原料去离子 水和人溶菌酶溶液， 搅拌至 40°C时停机出料，（在符合 GMP要求的制药工厂， 按制药规程 完 制成面霜。 V 2. Add the raw material glycerin, emulsifier HR-S, hyaluronic acid HA, Jieqiongyi II and deionized water to the emulsification pot. Heat and stir to 87-89 Ό; 3. Start the vacuum pump and vacuum the emulsifier to 0. 06Mpa, stop the vacuum, pump the oil phase into the emulsifier pot for 10 minutes at high speed, stir at 70 rpm, stop at high speed and homogenize; Homogenize for 10 minutes, stop homogenization, continue to stir for 10 minutes, the rotation speed is 60 rpm; 4. Slow cooling with water, add the raw thickener SepiGEL305 and the anti-itching agent SM- 650 for 2 minutes after the temperature is 60 Ό. Vacuum degassing; 5, when adding 45 ° C raw material deionized water and human lysozyme solution, stop to discharge when stirring to 40 ° C, (in a GMP-compliant pharmaceutical factory, according to the pharmaceutical regulations to complete the cream.
3、 乳液：  3. Emulsion:
配方： （重量份 ）  Recipe: (parts by weight)
( 1 ) 重组人溶菌酶 300万 U/mL/g  (1) Recombinant human lysozyme 3 million U/mL/g
(2) 二甲基硅油 200cst 2.0-5.0  (2) Dimethyl silicone oil 200cst 2.0-5.0
(3 ) 18醇 3.-5.0  (3) 18 alcohol 3.-5.0
(4) 辛碳癸酸甘油酯 /GTCC 2-5.0  (4) Cyanuric acid glyceride / GTCC 2-5.0
(5) 棕榈酸异辛酯 /GD— 5103 4.0-7.0  (5) Isooctyl palmitate /GD-5103 4.0-7.0
( 6) 维生素 E/VE 0.3-0.7  (6) Vitamin E/VE 0.3-0.7
(7 ) 乳化剂 Montanov 68 1.0-3.0  (7) Emulsifier Montanov 68 1.0-3.0
(8) 甘油 5-8  (8) Glycerin 5-8
(9) 乳化剂 HR—S! 1.0-4.0  (9) Emulsifier HR-S! 1.0-4.0
( 10) 透明脂酸 HA 0.01 -0.04  (10) Transparent fatty acid HA 0.01 -0.04
( 11 ) 杰琼一 II 1. 0-2. 0  (11) Jieqiongyi II 1. 0-2. 0
( 12) 香精 0.10-0.20  (12) Fragrance 0.10-0.20
(13)去离子水 70.0-76.0  (13) Deionized water 70.0-76.0
( 14) 杀螨止痒剂 SM- 650  ( 14) Killing and anti-itching agent SM- 650
生产工艺： 1、 将原料二甲基硅油 200cst、 18醇、 辛碳癸酸甘油酯 /GTCC、 棕榈酸异 辛酯 /GD— 5103、 维生素 E/VE、 乳化剂 Montanov 68 加入油相化料锅中， 搅拌加热到 85 °C; 2、 将原料甘油、 乳化剂 HR— Si、 透明脂酸 HA、 杰琼一 II和去离子水加至乳化锅中 加热搅拌至 87-89°C; 3、 启动真空泵将乳化锅抽真空到 0.06Mpa， 停止真空， 将油相抽进 乳化锅内高速均质 10分钟， 搅拌转速 70转， 高速均质后停止； 低速均质 10分钟， 停止均 质，继续搅拌 10分钟，转速为 60转； 4、通水慢速冷却，温度 60°C时加入杀螨止痒剂 SM- 650 均质 2分钟后真空脱气； 5、 当 45°C时加入原料去离子水和人溶菌酶溶液， 搅拌至 40°C时 停机出料,（在符合 GMP要求的制药工厂， 按制药规程完成） 制成乳液。 Production process: 1. Add raw material dimethyl silicone oil 200cst, 18 alcohol, octyl carbonic acid glyceride / GTCC, isooctyl palmitate / GD - 5103, vitamin E / VE, emulsifier Montanov 68 into the oil phase chemical pot Medium, stir and heat to 85 °C; 2, the raw material glycerin, emulsifier HR-Si, hyaluronic acid HA, Jieqiong II and deionized water are added to the emulsifier pot and heated to 87-89 ° C; 3, start the vacuum pump to pump the emulsification pot Vacuum to 0.06Mpa, stop the vacuum, pump the oil phase into the emulsifier pot for high-speed homogenization for 10 minutes, stir the rotation speed for 70 rpm, stop at high speed and homogenize; stop at low speed for 10 minutes, stop homogenization, continue stirring for 10 minutes, the rotation speed is 60 rpm; 4, slow cooling through water, temperature 60 ° C, add anti-itch agent SM- 650 homogenization 2 minutes after vacuum degassing; 5, when adding 45 ° C raw material deionized water and human lysozyme The solution is stopped when it is stirred to 40 ° C. (In a pharmaceutical factory that meets GMP requirements, it is completed according to the pharmaceutical regulations).
4、 面膜：  4, mask:
配方： （重量份） Recipe: (parts by weight)
(1) 重组人溶菌酶 300万 U/mL/g  (1) Recombinant human lysozyme 3 million U/mL/g
(2) 卡波树脂 CP941 0.2-0.5  (2) Kappa resin CP941 0.2-0.5
(3) 羧甲基纤维素 CMC 0.3-0.6  (3) Carboxymethyl cellulose CMC 0.3-0.6
(4) 聚乙烯醇 1788 (PVA) 7-10  (4) Polyvinyl alcohol 1788 (PVA) 7-10
(5) 甘油 6-8  (5) Glycerin 6-8
(6) 吐温一 80 (T— 80) 0.5-1.5  (6) Tween 80 (T-80) 0.5-1.5
(7) 氮酮 0.3-1.0  (7) Azones 0.3-1.0
(8) 乙醇 5-10  (8) Ethanol 5-10
(9) 香精 0.1-0.5  (9) Fragrance 0.1-0.5
(10) 水 67.6-80.45  (10) Water 67.6-80.45
(11) 杰玛一 II 0.15-0.3  (11) Gemma II II 0.15-0.3
(12) 杀螨止痒剂 SM- 650 0.5—1  (12) Killing and anti-itching agent SM- 650 0.5-1
生产工艺： 1、 首先将原料卡波树脂 941和羧甲基纤维素 CMC均匀分散于甘油中， 在 搅拌情况下将离子水加入充分混均匀， 此过程应在生产前 4小时备好。 之后搅拌下加入原 料聚乙烯醇 1788。 2、 将搅拌均匀的 1加入乳化锅内， 然后加入吐温一 80、 氮酮和水溶性 硅油搅拌加热到 75°C， 启动均质， 高速均质 10分钟， 然后停止高速均质， 低速均质 15分 钟后停止， 再继续搅拌 10分钟， 搅拌转速为 60转 /分。 3、 通过冷却水使其慢速冷却， 当 温度达到 50°C加入乙醇和香精和杀螨止痒剂 SM- 650然后继续搅拌到冷却， 并在温度低于 45 °C时加入人溶菌酶和杰琼后均质 2分钟，之后进行真空脱气。搅拌到物料 40°C停机出料， 制备成治疗痤疮的面膜成品。（此膜剂型要在符合 GMP要求的制药工厂，按制药规程完成）。 Production process: 1. Firstly, the raw material Kappa resin 941 and carboxymethyl cellulose CMC are uniformly dispersed in glycerin, and the ionized water is fully mixed and mixed under stirring, and the process should be prepared 4 hours before production. The raw material polyvinyl alcohol 1788 was then added with stirring. 2. Add the evenly stirred 1 into the emulsification pot, then add Tween 80, azone and water-soluble silicone oil and stir to heat to 75 °C, start homogenization, homogenize at high speed for 10 minutes, then stop high-speed homogenization, low speed Quality 15 points After the clock was stopped, stirring was continued for another 10 minutes, and the stirring speed was 60 rpm. 3, through the cooling water to slow cooling, when the temperature reaches 50 ° C, add ethanol and flavor and killing anti-itch agent SM- 650 and then continue to stir to cool, and add human lysozyme and when the temperature is lower than 45 °C After Jieqiong homogenization for 2 minutes, vacuum degassing was carried out. Stir to the material at 40 ° C to stop the discharge, and prepare the finished mask for treating acne. (This film type should be completed in accordance with GMP regulations in pharmaceutical factories that meet GMP requirements).
5、 化妆水： 将纯度 95%〜99%的基因重组人溶菌酶制得 1500U〜30万 U/ml/g， 磷酸 盐缓冲液 10〜20mM(pH6. 0〜7. 5)75〜85%、 5〜 25%丙二醇或丙三醇、 5〜15% 2—吡咯垸 酮一 5—羧酸钠、万分之 1〜5吐温 80， 1〜5%水溶性氮酮、尼泊金 0. 01〜0. 05%、 山梨酸钾 0. 01〜0. 1%， 在常温下混合均质， 制成化妆水。  5, lotion: the purity of 95% ~ 99% of the recombinant human lysozyme to obtain 1500U ~ 300,000 U / ml / g, phosphate buffer 10 ~ 20mM (pH 6. 0 ~ 7. 5) 75 ~ 85% 5~ 25% propylene glycol or glycerol, 5~15% 2-pyrrolidone- 5-carboxylic acid sodium, 1/5 to 10% Tween 80, 1~5% water-soluble azone, paraben 0. 01〜0. 05%, potassium sorbate 0. 01~0. 1%, mixed and homogenized at room temperature, made into a lotion.
上述配方中的重组人溶菌酶由大连奇龙生物技术研究所提供， 其它原料由北京日化研 究所提供。  The recombinant human lysozyme in the above formula was provided by Dalian Qilong Biotechnology Research Institute, and other raw materials were provided by Beijing Rihua Research Institute.
为了更好地理解本发明的实质， 下面将用基因重组人溶菌酶的药理试验及结果来说明 其在化妆品领域中的针对痤疮的新用途。  In order to better understand the essence of the present invention, pharmacological tests and results of recombinant human lysozyme will be used to illustrate its new use in the field of cosmetics for acne.
基因重组人溶菌酶以配制 200毫升培养基为基准， 用 ¾P044— 8毫升 MgS04l -5 克， K2S042_6克， KOH1—3克， CaS042H201—3.5克， 加蒸馏水至 200毫升， 高压灭菌 后接种甘油管种子， 摇床转数为每分钟 250转， 培养温度为 20— 35°C， 在恒温床上培养 36 一 48小时。 进行种子罐培养后进行生产罐培养。 将发酵表达完成的培养液进行提取纯化， 对提取纯化的蛋白浓缩液冰冻干燥， 测定蛋白量、 纯度和重组人溶菌酶活性（30000U/mg) 保存。 Recombinant human lysozyme is prepared on the basis of 200 ml medium, using 3⁄4P0 4 4-8 ml MgS0 4 l -5 g, K 2 S0 4 2_6 g, KOH 1-3 g, CaS0 4 2H 2 01-3.5 g, plus Distilled water to 200 ml, inoculated with glycerol tube seeds after autoclaving, the number of revolutions of the shaker was 250 rpm, the culture temperature was 20-35 ° C, and the culture was carried out for 36 to 48 hours on a constant temperature bed. Production tank culture is carried out after seed tank cultivation. The culture solution in which the fermentation expression was completed was extracted and purified, and the extracted and purified protein concentrate was freeze-dried, and the amount of protein, purity, and recombinant human lysozyme activity (30000 U/mg) were measured and stored.
一、 基因重组人溶菌酶 （HLZ) 体外抗菌作用评价 I. Evaluation of in vitro antibacterial activity of recombinant human lysozyme (HLZ)
药品及试剂 Drugs and reagents
1、 基因重组人溶菌酶浓缩液 （Human Lysozyme HLZ) 活性单位： 30000单位 /mL， 由大连奇龙生物技术研究所提供，  1. Human recombinant Lysozyme HLZ Active unit: 30000 units / mL, provided by Dalian Qilong Biotechnology Research Institute,
2、对照溶菌酶（ contral Lysozyme, CLZ): 白色粉未， 活性单位： 50000单位 /mg， 美国 SIGMA公司产品， 批号： L6876。 3、 克拉霉素： 效价 948 /mg， 中国药品生物制品检定所标准品， 批号：2, control lysozyme (Contra Lysozyme, CLZ): white powder, active unit: 50000 units / mg, the United States SIGMA company products, batch number: L6876. 3, clarithromycin: potency 948 / mg, China National Institute for the Control of Pharmaceutical and Biological Products, batch number:
4、 罗红霉素： 效价 878 u /mg， 中国药品生物制品检定所标准品， 批号-4. Roxithromycin: Potency 878 u /mg, China National Institute for the Control of Pharmaceutical and Biological Products, batch number -
5、 注射用阿莫西林： 哈尔滨制药厂产品， 批号： 010504。 5. Amoxicillin for injection: Harbin Pharmaceutical Factory, batch number: 010504.
6、 琼脂糖 (B10WEST AGAROSE):  6, agarose (B10WEST AGAROSE):
7、 三羟甲基氨基甲垸 （Tris): 成都化学试验厂， 批号 010211  7. Tris (Tris): Chengdu Chemical Testing Plant, batch number 010211
试验菌株 Test strain
所用菌株均为 2002. 4〜2003. 9月于四川、 北京地区收集的临床分离致病菌。 经本室 用 API方法重新鉴定后用于试验。  The strains used were all clinical isolates collected in Sichuan and Beijing in 2002. 4~2003. It was re-identified by the API method and used for testing.
质控菌株： 金黄色葡萄球菌 ATCC25923 Quality Control Strain: Staphylococcus aureus ATCC25923
肠埃希菌 ATCC25922  Escherichia coli ATCC25922
铜绿假单孢菌 ATCC27853  Pseudomonas aeruginosa ATCC27853
养基：  Nutrient:
1、 Tris- HC1缓冲液： 0. 1M Tris 100ml , 0. 1M HC1 70ml , High Water 800ml , 测 PH值， 用 HC1溶液调至 PH7. 2， 加 High Water至 1000ml。  1. Tris-HC1 buffer: 0. 1M Tris 100ml, 0. 1M HC1 70ml, High Water 800ml, measure PH value, adjust to pH7.2 with HC1 solution, add High Water to 1000ml.
2、 Tris-HCl脂琼养基： 在 Tris-Cl缓冲液中加入脂琼糖 116°C无菌后使用。  2. Tris-HCl lipid agar base: Add the lipid agarose in Tris-Cl buffer and use it at 116 °C.
3、 M- H培养基： 中国药品生物制品检定所产品， M-H肉汤培养基： 称取 25g加 1000ml 蒸馏水，加热溶解，分装，高压灭菌， 116 20分钟。 M-H固体培养基：称取 36g，加 1000ml 蒸馏水， 高压灭菌， 116Ό 20分钟,用于革兰阳性、 阴性需氧菌的药敏试验。  3, M-H medium: China National Institute for the Control of Pharmaceutical and Biological Products, M-H broth culture medium: Weigh 25g and 1000ml distilled water, heat to dissolve, dispense, autoclave, 116 20 minutes. M-H solid medium: Weigh 36g, add 1000ml of distilled water, autoclave, 116Ό 20 minutes, for the susceptibility test of Gram-positive, negative aerobic bacteria.
4、 血培养基， 即在 M- H培养基中加 5-10%脱纤维兔血配制而成， 用于肠球菌、 链球 菌的药敏试验。  4. Blood culture medium, which is prepared by adding 5-10% defibrated rabbit blood to M-H medium, for drug sensitivity test of Enterococcus and Streptococcus.
试验方法： experiment method:
采用琼脂二倍稀释法测定受试药物对试验菌株的最低抑菌浓度 （MIC)。 将受试药物 用灭菌蒸镏水溶解， 适当稀释。 分别取 lml药液加 9ral 融化的 Tris- HC1琼脂糖固体培养 基混匀， 以二倍稀释，制备系列含药平皿。每皿所含药物终浓度分别为 4、 2、 1、 0. 5、 0. 25、 0. 125、 0. 06、 0. 03…… 0. OOlmg/ml ; 用 多点接种仪 (Denley A400, England) 将稀释 至 105CFU/ml的试验菌液接种于各含药平皿表面，置于 37Ό培养 8-10小时，取出分别吸取 6ml融化的 M- H培养基（50Ό )覆盖上述系列平皿表面， 再置于 37Ό培养 10小时取出， 观 察结果， 以无细菌生长平皿内所含最低药物浓度为该菌的最低抑菌浓度 （MIC；)。 试验结果 （1 ) 人溶菌酶对临床分离菌株的体外抗菌活性见表 1。 (2) 人溶菌酶对 379 株临床分离致病菌的 MIC50、 MIC90见表 1。 根据以上试验结果表明： 人溶菌酶体外具有一定抗菌作用， 对耐药菌株的抗菌活性也 有很好的作用。 人溶菌酶是一种小分子蛋白质， 存在于机体的泪液、 痰、 鼻涕、 白细胞和 血清中， 对多种革兰氏阳性菌和少数革兰氏阴性菌有杀菌作用， 基因重组人溶菌酶（Human The minimum inhibitory concentration (MIC) of the test drug against the test strain was determined by agar double dilution method. The test drug is dissolved in sterile distilled water and diluted appropriately. The lcc solution was mixed with 9 ml of melted Tris-HC1 agarose solid medium, and diluted twice to prepare a series of drug-containing plates. The final concentration of the drug contained in each dish is 4, 2, 1, 0.5, 0.52, respectively. 0. 125, 0. 06, 0. 03...... 0. OOlmg/ml; a test solution diluted to 10 5 CFU/ml was inoculated onto the surface of each drug-containing plate using a multi-point inoculation instrument (Denley A400, England). After being placed in 37 Ό for 8-10 hours, take out 6 ml of melted M-H medium (50 Ό) to cover the surface of the above-mentioned series of dishes, and then place them in 37 Ό for 10 hours to observe, and observe the results, and the lowest in the bacteria-free growth plate. The drug concentration is the minimum inhibitory concentration (MIC;) of the bacteria. Test results (1) The in vitro antibacterial activity of human lysozyme on clinical isolates is shown in Table 1. (2) The MIC50 and MIC90 of human lysozyme against 379 clinical isolates of pathogenic bacteria are shown in Table 1. According to the above test results, human lysozyme has a certain antibacterial effect in vitro, and has a good effect on the antibacterial activity of the resistant strain. Human lysozyme is a small molecule protein found in the body's tears, sputum, sputum, white blood cells and serum. It has a bactericidal effect on a variety of Gram-positive bacteria and a few Gram-negative bacteria. Recombinant human lysozyme ( Human
Lysozyme)主要切断肽聚糖中 N-乙酰葡萄糖胺和 N-乙酰胞壁酸之间的 β _1、 4糖苷键之间 的联结， 破坏肽聚糖支架， 引起细菌裂解。 Lysozyme) mainly cuts off the linkage between the β _1 and 4 glycosidic bonds between N-acetylglucosamine and N-acetylmuramic acid in peptidoglycan, destroys the peptidoglycan scaffold and causes bacterial lysis.
表 1 人溶菌酶、 鸡溶菌酶、 克拉霉素和罗红霉素体外抗菌活性  Table 1 In vitro antibacterial activity of human lysozyme, chicken lysozyme, clarithromycin and roxithromycin
MIC  MIC
细菌 CLZ  Bacteria CLZ
HLZ CLA ROX  HLZ CLA ROX
mg/ml (万 μ  Mg/ml (million μ
mg/ml (万 μ /ml) mg/ml mg/ml  Mg/ml (10,000 μg/ml) mg/ml mg/ml
/ml)  /ml)
金葡 MRSA02-22 0.025(0.03) 0.008(0.04) >1 〉1 金葡 MRSA02-23 0.025(0.03) 0.008(0.04) >1 >1 金葡 MRSA02-26 0.025(0.03) 0.004(0.02) >1 >1 金葡 MRSA02-28 0.5(1.5) 0.016(0.08) >1 >1 金葡 02-19-5 0.03(0.1) <0.002(0.001) >1 >1 表葡 MssE25 0.016(0.05) 0.004(0.02) <0.001 <0.001 表葡 MRSE  Gold Portuguese MRSA02-22 0.025(0.03) 0.008(0.04) >1 〉1 Golden Portuguese MRSA02-23 0.025(0.03) 0.008(0.04) >1 >1 Golden Portuguese MRSA02-26 0.025(0.03) 0.004(0.02) >1 > 1 Golden Portuguese MRSA02-28 0.5(1.5) 0.016(0.08) >1 >1 Golden Portuguese 02-19-5 0.03(0.1) <0.002(0.001) >1 >1 Table Portuguese MssE25 0.016(0.05) 0.004(0.02) < 0.001 <0.001 table of MRSE
0.063(0.2) 0.5(2.5) 0.03 0.5 02-29  0.063(0.2) 0.5(2.5) 0.03 0.5 02-29
表葡 MRSE 02-5 <0.001(0.003) <0.001(0.003) <0.001 <0.001 表葡 MRSE 02-6 0.001(0.005) 0.001(0.005) <0.001 <0.001 表葡 MRSE  Table MR MRSE 02-5 <0.001 (0.003) <0.001 (0.003) <0.001 <0.001 Table Portuguese MRSE 02-6 0.001 (0.005) 0.001 (0.005) <0.001 <0.001 Table Portuguese MRSE
<0.001(0.003) 〈0.001(0.005) >1 1
Figure imgf000011_0001
<0.001 (0.003) <0.001 (0.005) >1 1
Figure imgf000011_0001
Λ Λ Λ  Λ Λ Λ
< Λ Λ  < Λ Λ
0.00004.12II V V 丙酸杆菌 02-7-4 0.008(0.02) 4(20) 0.002 0.002 丙酸杆菌 02-7-5 0.008(0.02) 0.25(0.63) <0.001 <0.001 丙酸杆菌 02-7-6 0.008(0.02) 0.063(0.31) 0.008 0.25 丙酸杆菌 02-7-9 0.063(0.2) 0.125(0.63) 0.016 丙酸杆菌 02-7-7 0.125(0.4) 0.016(0.08) 0.032 0.25 丙酸杆菌 03-2-22 0.032(0.1) 0.5(2.5) 1 1 丙酸杆菌 03-2-30 0.063(0.2) 4(10) >1 >1 丙酸杆菌 03-2-32 0.016(0.05) 0.25(1.25) 0.008 0.5 丙酸杆菌 03-2-33 0.032(0.1) 2(10) >1 >1 丙酸杆菌 03-2-36 0.063(0.2) 4(20) 0.25 0.5 丙酸杆菌 03-2-37 0.032(0.1) >4(20) >1 1 丙酸杆菌 03-2-39 0.032(0.1) 0.5(2.5) 0.5 1 续表 1 0.00004.12II VV Propionibacterium 02-7-4 0.008(0.02) 4(20) 0.002 0.002 Propionibacterium 02-7-5 0.008(0.02) 0.25(0.63) <0.001 <0.001 Propionibacterium 02-7-6 0.008(0.02) 0.063(0.31) 0.008 0.25 Propionibacterium 02-7-9 0.063(0.2) 0.125(0.63) 0.016 Propionibacterium 02-7-7 0.125(0.4) 0.016(0.08) 0.032 0.25 Propionibacterium 03-2-22 0.032 (0.1) 0.5(2.5) 1 1 Propionibacterium 03-2-30 0.063(0.2) 4(10) >1 >1 Propionibacterium 03-2-32 0.016(0.05) 0.25(1.25) 0.008 0.5 Propionibacterium 03-2-33 0.032(0.1) 2(10) >1 >1 Propionibacterium 03-2-36 0.063(0.2) 4(20) 0.25 0.5 Propionibacterium 03-2-37 0.032(0.1) >4( 20) >1 1 Propionibacterium 03-2-39 0.032(0.1) 0.5(2.5) 0.5 1 Continued Table 1
o  o
MIC o  MIC o
o  o
HLZ CLZ  HLZ CLZ
细菌 CLA ROX mg/ml (万 μ mg/ml (万  Bacteria CLA ROX mg/ml (10,000 μmg/ml (10,000
mg/ml mg/ml /ml) /ml)  Mg/ml mg/ml /ml) /ml)
大肠埃希菌 03-2-41 0.032(0.1) 0.5(2.5) >1 〉1 大肠埃希菌 03-2-42 0.016(0.05) 0.5(2.5) 0.063 >1 大肠埃希菌 03-2-43 <0.001(0.003) >4(20) 0.016 0.25 大肠埃希菌 03-2-45 0.032(0.1) 2(10) 0.25 0.5 大肠埃希菌 03-2-51 0.032(0.1) >4(20) >1 >1 大肠埃希菌 03-2-52 0.032(0.1) >4(20) >1 0.25 大肠埃希菌 03-2-53 0.008(0.024) 4(20) 0.03 0.25 大肠埃希菌 032-54 0.032(0.1) 1(5) >1 1 大肠埃希菌 03-2-56 0.032(0.1) 〉4(20) 0.016 0.25 大肠埃希菌 03-2-57 <0.001(0.003) 2(10) 0.032 0.25 大肠埃希菌 03-2-58 0.001(0.003) 4(20) >1 0.5 大肠埃希菌 03-2-59 0.032(0.1) 4(20) 0.125 0.25 沙雷氏菌 2-31-8 0.008(0.02) 0.5(0.25) 0.008 0.016 沙雷氏菌 2-31-8 0.008(0.02) 1(5) 0.008 0.016 沙雷氏菌 2-31-8 0.008(0.02) 0.125(0.63) 0.016 0.016 肺炎克雷伯菌 02-6-14 0.25(0.8) 1(5) 0.063 >1 肺炎克雷伯菌 02-6-18 0.5(1.5) >4(20) 0.5 1 注： HLZ指人溶菌酶， CLZ指对照溶菌酶（鸡溶菌酶）， CLA指克拉霉素， ROX指罗红霉 素 二、 重组人溶菌酶 （HLZ) 加杀螨止痒剂 SM-650体外杀螨作用评价 1、 基因重组人溶菌酶浓缩液活性单位： 300单位 /mL试验 药品及试剂 Escherichia coli 03-2-41 0.032(0.1) 0.5(2.5) >1 〉1 Escherichia coli 03-2-42 0.016(0.05) 0.5(2.5) 0.063 >1 Escherichia coli 03-2-43 <0.001(0.003) >4(20) 0.016 0.25 Escherichia coli 03-2-45 0.032(0.1) 2(10) 0.25 0.5 Escherichia coli 03-2-51 0.032(0.1) >4(20) > 1 >1 Escherichia coli 03-2-52 0.032(0.1) >4(20) >1 0.25 Escherichia coli 03-2-53 0.008(0.024) 4(20) 0.03 0.25 Escherichia coli 032-54 0.032(0.1) 1(5) >1 1 Escherichia coli 03-2-56 0.032(0.1) 〉4(20) 0.016 0.25 Escherichia coli 03-2-57 <0.001(0.003) 2(10) 0.032 0.25 Escherichia coli 03-2-58 0.001(0.003) 4(20) >1 0.5 Escherichia coli 03-2-59 0.032(0.1) 4(20) 0.125 0.25 Serratia 2-31-8 0.008( 0.02) 0.5(0.25) 0.008 0.016 Serratia 2-31-8 0.008(0.02) 1(5) 0.008 0.016 Serratia 2-31-8 0.008(0.02) 0.125(0.63) 0.016 0.016 Klebsiella pneumonia Bacteria 02-6-14 0.25(0.8) 1(5) 0.063 >1 Klebsiella pneumoniae 02-6-18 0.5(1.5) >4(20) 0.5 1 Note: HLZ refers to human lysozyme, CLZ refers to control lysate Enzyme (chicken lysozyme), CLA means clarithromycin, ROX means roxithromycin II, recombinant human lysozyme (HLZ) plus killing and anti-itching agent SM-650 in vitro killing effect evaluation 1, recombinant human lysozyme concentration Liquid activity unit: 300 units/mL test drug and reagent
1、 基因重组人溶菌酶浓缩液 （Human Lysozyme HLZ) 活性单位： 300 单位 /mL， 由 大连奇龙生物技术研究所提供， 1. Human recombinant Lysozyme HLZ Active unit: 300 units / mL, provided by Dalian Qilong Biotechnology Research Institute,
2、 杀螨止痒剂 SM- 650: 白色粉未， 纯度为 99%, ph : 6〜8, 生产单位： 北京耐特生 化技术研究所 试验螨虫： 所用螨虫标本为第四军医大学临床门诊痤疮病人。 试验方法： 体外杀螨虫活性测定： 釆用图片法测定了人溶菌酶 （HLZ) 加杀螨止痒剂 SM-650 最 低杀螨浓度 （MIC)。 将螨虫标本分离到玻片上， 用人溶菌酶 （HLZ) 300 单位 /mL， 杀螨止 痒剂 SM- 650 1%/Ml o 滴到玻片螨虫标本上， 观察结果， 以最低杀螨浓度 （MIC)。 试验结果见表 2， 结果表明人溶菌酶加杀螨止痒剂 SM-650体外杀螨有杀灭作用。 表 2 2. Killing and antipruritic agent SM-650: White powder, purity 99%, ph: 6~8, Production unit: Beijing Naite Biochemical Technology Research Institute test aphid: The aphid specimen used is the clinical outpatient hemorrhoid of the Fourth Military Medical University patient. Test method: Determination of in vitro aphid activity: 人 The human lysozyme (HLZ) plus the anti-itching agent SM-650 minimum killing concentration (MIC) was determined by photo method. The aphid specimens were separated onto a slide, and the human lysozyme (HLZ) 300 units/mL, the anti-itching agent SM-650 1%/Ml o was dropped onto the slide aphid specimens, and the results were observed to the minimum acaricidal concentration (MIC). ). The test results are shown in Table 2. The results indicate that human lysozyme plus acaricidal antipruritic SM-650 has a killing effect on in vitro killing. Table 2
螨虫标本 对照组 杀螨止痒剂 人溶菌酶加杀  Aphid specimens control group killing and antipruritic agent human lysozyme plus kill
SM-650 螨 止 痒 剂  SM-650 止 Antipruritic
SM-650  SM-650
1 号 12个螨 0 51% 62% 2号 18个螨 0 48% 59% No. 1 12 螨 0 51% 62% No. 2 18 螨 0 48% 59%
虫  Worm
3号 16个螨 0 49% 61%  No. 3 16 螨 0 49% 61%
虫  Worm
4号 11 个螨 0 50% 65%  No. 4 11 螨 0 50% 65%
虫  Worm
5 号 19个螨 0 53% 66%  No. 5 19 螨 0 53% 66%
虫  Worm
2、 基因重组人溶菌酶浓缩液活性单位： 3000单位 /mL试验 药品及试剂 2. Recombinant human lysozyme concentrate active unit: 3000 units / mL test drugs and reagents
1、 基因重组人溶菌酶浓缩液 （Human Lysozyme HLZ) 活性单位： 3000单位 /mL， 由 大连奇龙生物技术研究所提供， 1. Human recombinant Lysozyme HLZ Active unit: 3000 units / mL, provided by Dalian Qilong Biotechnology Research Institute,
2、 杀螨止痒剂 SM-650: 白色粉未， 纯度为 99 % , ph : 6〜8， 生产单位： 北京耐特生 化技术研究所 ' 试验螨虫： 所用螨虫标本为第四军医大学临床门诊痤疮病人。 试验方法： 体外杀螨虫活性测定： 采用图片法测定了人溶菌酶 （HLZ) 加杀螨止痒剂 SM-650 最 低杀螨浓度 （MIC)。 将螨虫标本分离到玻片上， 用人溶菌酶 （HLZ) 3000单位 /mL, 杀螨止 痒剂 SM- 650 1%/Ml c 滴到玻片螨虫标本上， 观察结果， 以半数杀螨浓度 （MBC)。 试验结果见表 3 , 结果表明人溶菌酶加杀螨止痒剂 SM-650体外杀螨有杀灭作用。 表 3 2. Killing and antipruritic agent SM-650: White powder, purity 99%, ph: 6~8, Production unit: Beijing Naite Biochemical Technology Institute' Test aphid: The aphid specimen used is the clinical clinic of the Fourth Military Medical University. Acne patients. Test method: In vitro aphid activity assay: The lowest killing concentration (MIC) of human lysozyme (HLZ) plus acaricidal antipruritic SM-650 was determined by photo method. The aphid specimens were separated onto the slides, and the human lysozyme (HLZ) 3000 units/mL, the anti-itching agent SM-650 1%/Ml c was dropped onto the slides of the aphid specimens, and the results were observed to obtain half the acaricidal concentration (MBC). ). The test results are shown in Table 3. The results show that human lysozyme plus acaricidal antipruritic SM-650 has a killing effect on in vitro killing. table 3
螨虫标本 对照组 杀螨止痒剂 人溶菌酶加杀  Aphid specimens control group killing and antipruritic agent human lysozyme plus kill
SM-650 螨 止 痒 剂  SM-650 止 Antipruritic
SM-650  SM-650
1 号 12个螨 0 730% 85%  No. 1 12 螨 0 730% 85%
虫  Worm
2 号 18 个螨 0 82% 91%  No. 2 18 螨 0 82% 91%
虫  Worm
3号 16个螨 0 80% 94%  No. 3 16 螨 0 80% 94%
虫  Worm
4号 11 个螨 0 76% 88%  No. 4 11 螨 0 76% 88%
虫  Worm
5 号 19个螨 0 79% 90% L ¾ I I I I No. 5 19 螨 0 79% 90% L 3⁄4 IIII
3、 基因重组人溶菌酶浓缩液活性单位： 30000单位 /mL试验 药品及试剂  3, recombinant human lysozyme concentrate activity unit: 30000 units / mL test drugs and reagents
1、 基因重组人溶菌酶浓缩液 （Human Lysozyme HLZ) 活性单位： 30000 单位 /mL， 由大连奇龙生物技术研究所提供， 1. Human recombinant Lysozyme HLZ Active unit: 30000 units / mL, provided by Dalian Qilong Biotechnology Research Institute,
2、 杀螨止痒剂 SM-650: 白色粉未， 纯度为 99%, ph: 6〜8, 生产单位： 北京耐特生 化技术研究所 试验螨虫： 所用螨虫标本为第四军医大学临床门诊痤疮病人。 试验方法： 体外杀螨虫活性测定： 采用图片法测定了人溶菌酶 （HLZ) 加杀螨止痒剂 SM-650 最 低杀螨浓度 （MIC)。 将螨虫标本分离到玻片上， 用人溶菌酶 （HLZ) 30000单位 /mL， 杀螨 止痒剂 SM- 650 1%/Ml o 滴到玻片螨虫标本上， 观察结果， 以无活螨虫为杀螨浓度。 试验结果见表 4， 结果表明人溶菌酶加杀螨止痒剂 SM- 650体外杀螨有杀灭作用。 表 4 2. Killing and antipruritic agent SM-650: White powder, purity 99%, ph: 6~8, Production unit: Beijing Naite Biochemical Technology Research Institute test aphid: The aphid specimen used is the clinical outpatient hemorrhoid of the Fourth Military Medical University. patient. Test method: In vitro aphid activity assay: The lowest killing concentration (MIC) of human lysozyme (HLZ) plus acaricidal antipruritic SM-650 was determined by photo method. The aphid specimens were separated onto a slide, and human lysozyme (HLZ) was used at 30,000 units/mL, and the anti-itching agent SM-650 1%/Ml o was dropped onto the slide aphid specimens. The results were observed, and the live aphids were killed. concentration. The test results are shown in Table 4. The results show that human lysozyme plus acaricidal antipruritic SM-650 has a killing effect on in vitro killing. Table 4
Figure imgf000015_0001
Figure imgf000015_0001
采用图片法测定了人溶菌酶 （HLZ) 加杀螨止痒剂 SM-650对临床分离五人份的螨虫标 本体外杀螨作用， 结果表明： 人溶菌酶加杀螨止痒剂 SM- 650对螨虫有杀灭作用。杀螨止痒 剂 SM-650最低杀螨浓度 MICRi ^为 0. 2%~2%o人溶菌酶（HLZ)活性单位： 300单位 /ml~30000 万单位 /ml。 由大连奇龙生物技术研究所研制的基因重组人溶菌酶（Human Lysozyme)具有 其它溶菌酶相同作用机制， 主要切断肽聚糖中 N-乙酰葡萄糖胺和 N-乙酰胞壁酸之间的 e The in vitro killing effect of human lysozyme (HLZ) plus acaricidal antipruritic SM-650 on five clinical isolates of aphid samples was determined by photo method. The results showed that: human lysozyme plus acaricidal antipruritic SM-650 pair Aphids have a killing effect. The minimum acaricidal concentration of the killing and antipruritic agent SM-650 is MIC Ri ^ 0. 2% ~ 2% o human lysozyme (HLZ) activity unit: 300 units / ml ~ 300 million units / ml. The recombinant human lysozyme (Human Lysozyme) developed by Dalian Qilong Biotechnology Research Institute has Other lysozymes have the same mechanism of action, mainly cutting off the e between the N-acetylglucosamine and the N-acetylmuramic acid in the peptidoglycan.
-1、 4糖苷键之间的联结， 破坏肽聚糖支架， 引起细菌裂解。 杀螨止痒剂 SM-650产品有很 强的杀螨作用。 三、 人溶菌酶对巴豆油诱发小鼠耳廓肿胀的影响 选用体重 27-30克的健康雄性昆明小鼠 50只，动物自由饮水，试验室温度控制在 22-28 °C左右， 动物喂饲大鼠标准饲料。随机分成 5组，每组 10只。第一组为空白对照组； 第二、 三、 四组为人溶菌酶， 剂量分别为 120、 60、 30IU/只； 第五组为鸡溶菌酶 （阳性对照）， 30IU/只。 首先， 各组小鼠全部右耳内侧涂 1%巴豆油 （巴豆油， 浅棕色油状液体， 药用级， 批号 000309, 由江西吉水县华宝天然药用厂生产。 临用前用乙醇： 水： 乙醚 25: 5： 70混 合溶媒配制成 1%浓度备用。） 30μί致炎，半小时后分别用双蒸水 20μ1、人溶菌酶（6000IU/ML) 20μ1、人溶菌酶 ( 3000IU/ML) 20μΐ、人溶菌酶 ( 1500IU/ML) 20μ1、鸡溶菌酶 ( 1500IU/ML) 20μ1涂抹各组小鼠右耳内侧。致炎后 4小时将小鼠脱颈椎致死， 沿耳廓基线剪下左右两耳 片， 用 8讓打孔器冲下两耳片， 精确称重， 左右耳片重量之差为肿胀度。 经 Τ检验比较药 物组与空白对照组的差异， 并求出抑制率。 试验重复一次。 试验结果见表 17-22。 小鼠经外涂人溶菌酶 120、 60、 30IU/只， 使小鼠由巴豆油诱发 耳廓肿胀度明显减轻， 经 Τ检验， 药物组与空白对照组比较， 有显著性差异（Ρ<0. 05 )， 说 明人溶菌酶具有抗炎作用。 · The linkage between the -1 and 4 glycosidic bonds disrupts the peptidoglycan scaffold and causes bacterial lysis. Killing and anti-itching agent SM-650 has a strong killing effect. Effects of human lysozyme on the swelling of mouse ear-induced ear edema 50 healthy male Kunming mice weighing 27-30 grams were used, the animals were free to drink water, and the temperature of the laboratory was controlled at 22-28 °C. Animal feeding Rat standard feed. Randomly divided into 5 groups of 10 each. The first group was a blank control group; the second, third and fourth groups were human lysozyme at doses of 120, 60, 30 IU/head; the fifth group was chicken lysozyme (positive control), 30 IU/only. First, all groups of mice were coated with 1% croton oil (croton oil, light brown oily liquid, pharmaceutical grade, batch number 000309, produced by the Huabao Natural Medicine Factory of Jishui County, Jiangxi Province). : Ether 25: 5: 70 mixed solvent is prepared to make 1% concentration for use.) 30μί inflammation, after half an hour, use double distilled water 20μ1, human lysozyme (6000IU/ML) 20μ1, human lysozyme (3000IU/ML) 20μΐ Human lysozyme (1500 IU/ML) 20 μl, chicken lysozyme (1500 IU/ML) 20 μl was applied to the inner side of the right ear of each group of mice. The mice were sacrificed from the cervical vertebrae 4 hours after the inflammation. The left and right ears were cut along the baseline of the auricle. The punches were used to punch the two ears and accurately weighed. The difference between the left and right ear weights was the degree of swelling. The difference between the drug group and the blank control group was compared by the sputum test, and the inhibition rate was determined. The test was repeated once. The test results are shown in Table 17-22. The mice were exposed to human lysozyme 120, 60, 30 IU/only, which significantly reduced the degree of ear swelling caused by croton oil in mice. After the sputum test, there was a significant difference between the drug group and the blank control group (Ρ<0 05), indicating that human lysozyme has an anti-inflammatory effect. ·
表 17-22 人溶菌酶对巴豆油诱发小鼠耳廓肿胀的影响  Table 17-22 Effect of human lysozyme on ear swelling of mice induced by croton oil
第 1次试验 第 2次试验  First trial second trial
组别 剂量  Group dose
( IU/只） 肿胀度 抑制率 肿胀度 抑制率  (IU/only) swelling degree inhibition rate swelling degree inhibition rate
(mg, X土 SD) (%) (mg, X±SD) (%)  (mg, X soil SD) (%) (mg, X±SD) (%)
空白对照 ― 20. 3 ± 3. 40 20. 1 ±4. 01  Blank control ― 20. 3 ± 3. 40 20. 1 ±4. 01
人溶菌酶 120 12. 5 ±5. 56** 38. 42 13. 6± 3. 78** 32. 34  Human lysozyme 120 12. 5 ±5. 56** 38. 42 13. 6± 3. 78** 32. 34
人溶菌酶 60 13. 5±4. 67** 33. 50 15. 1 ±2. 42** 24. 88  Human lysozyme 60 13. 5±4. 67** 33. 50 15. 1 ±2. 42** 24. 88
人溶菌酶 30 14. 2 ± 3. 77** 30. 05 15. 4±4. 22* 23. 38  Human lysozyme 30 14. 2 ± 3. 77** 30. 05 15. 4±4. 22* 23. 38
鸡溶菌酶 30 14. 6±2. 12** 28. 08 15. 1 ±2. 51** 24. 88  Chicken lysozyme 30 14. 6±2. 12** 28. 08 15. 1 ±2. 51** 24. 88
注： 与空白对照组比较， *P<0. 05， **P<0. 01. 本发明对基因重组人溶菌酶发掘了新的用途， 开拓了一个新的应用领域。 由重组人溶 菌酶制得的化妆品安全无毒副作用， 有很好的应用前景。 基因重组人溶菌酶来源丰富， 制 备工艺简单， 并可做成洗面奶或洁面乳、 香皂、 化妆水、 乳液、 面霜或面膜等， 使用方便， 和现有各种化妆品使用方法相同， 可以构成系列化妆品成套使用。 Note: Compared with the blank control group, *P<0.05, **P<0. 01. The invention has opened up a new application for genetic recombinant human lysozyme and opened up a new application field. The cosmetics prepared by recombinant human lysozyme have safe and non-toxic side effects and have good application prospects. Recombinant human lysozyme is rich in source, simple in preparation process, and can be used as facial cleanser or cleansing lotion, soap, lotion, lotion, cream or mask. It is easy to use and can be used in the same way as various existing cosmetics. Cosmetics are used in sets.
具体实施方式： detailed description:
下面结合实施例对本发明做进一步的描述：  The present invention will be further described below in conjunction with the embodiments:
实施例 1 : Example 1
基因重组人溶菌酶以配制 200毫升培养基为准， 用磷酸 6毫升、 硫酸镁 3克， 硫酸钾 4克， 氢氧化钾 1克， 硫酸钙 1.5克， 加蒸馏水至 200毫升， 高压灭菌后接种甘油管种子， 摇床转数为每分钟 250转， 培养温度为 20— 35°C， 在恒温床上培养 36— 48小时。 进行种 子罐培养， 最后进行生产罐培养。 将发酵表达完成的培养液进行提取纯化， 对提取纯化的 基因重组人溶菌酶浓缩液进行测蛋白、 测活性保存。 将纯度 95%基因重组人溶菌酶浓缩液 溶于水中制得 30000U/mL备用。  Recombinant human lysozyme is prepared by preparing 200 ml of medium, using 6 ml of phosphoric acid, 3 g of magnesium sulfate, 4 g of potassium sulfate, 1 g of potassium hydroxide, 1.5 g of calcium sulfate, and adding distilled water to 200 ml. After autoclaving. The glycerol tube seed was inoculated, the number of revolutions of the shaker was 250 rpm, the culture temperature was 20-35 ° C, and the culture was carried out for 36-48 hours on a constant temperature bed. The seed tank culture is carried out, and finally the production tank culture is carried out. The culture solution in which the fermentation expression is completed is extracted and purified, and the extracted and purified recombinant human lysozyme concentrate is subjected to protein measurement and activity storage. A 95% pure recombinant human lysozyme concentrate was dissolved in water to prepare 30000 U/mL for use.
将纯度 95%的基因重组人溶菌酶制得 30000 U/mL/g , 磷酸盐缓冲液 10〜 20mM(pH6. 5〜7. 5)80%、 15%丙二醇、 6% 2 —吡咯烷酮一 5—羧酸钠、 0.9%水溶性氮酮、 万 分之三吐温 80， 杀螨止痒剂 SM- 650纯度为 99%， ph: 6〜8 (商品） 1%， 在常温下混合 均质， 制成化妆水。  The recombinant human lysozyme having a purity of 95% was prepared to have a 30,000 U/mL/g, a phosphate buffer solution of 10 to 20 mM (pH 6.5 to 7.5), 80%, 15% propylene glycol, and 6% 2-pyrrolidone-5. Sodium carboxylate, 0.9% water-soluble azone, 3/10,000 Tween 80, killing and anti-itching agent SM-650 with a purity of 99%, ph: 6~8 (commodity) 1%, mixed at room temperature, Make a lotion.
实施例 2: Example 2:
按照实施例 1所述方法制备人溶菌酶， 人溶菌酶治疗痤疮护肤膏按下述配方及工艺生 产：  The human lysozyme was prepared according to the method described in Example 1. The human lysozyme treatment acne skin cream was produced according to the following formula and process:
配方： （重量）  Recipe: (weight)
( 1 ) 重组人溶菌酶 3万 U/mL/g  (1) Recombinant human lysozyme 30,000 U/mL/g
(2) 二甲基硅油 200cst 2.0  (2) Dimethyl silicone oil 200cst 2.0
(3 ) 18醇 3.0 (4) 辛碳癸酸甘油酯 /GTCC 2 (3) 18 alcohol 3.0 (4) Cyanuric acid glyceride / GTCC 2
(5) 棕榈酸异辛酯 /GD— 5103 4.0  (5) Isooctyl palmitate /GD-5103 4.0
(6) 维生素 E/VE 0.3  (6) Vitamin E/VE 0.3
(7) 乳化剂 Montanov 68 1.0  (7) Emulsifier Montanov 68 1.0
(8) 甘油 8  (8) Glycerin 8
(9) 乳化剂 HR—S! 1.0  (9) Emulsifier HR-S! 1.0
(10) 透明脂酸 HA 0.01  (10) Transparent fatty acid HA 0.01
(11) 增稠剂 SepiGEL305 0.1  (11) Thickener SepiGEL305 0.1
(12) 杰玛— II 1.0  (12) Gemma - II 1.0
(13) 香精 0.10  (13) Fragrance 0.10
(14)去离子水 71.  (14) Deionized water 71.
(15) 杀螨止痒剂 SM- 650 0.5  (15) Killing and anti-itching agent SM- 650 0.5
工艺： 1、将原料二甲基硅油 200cst、 18醇、辛碳癸酸甘油酯 /GTCC、棕榈酸异辛酯 /GD 一 5103、 维生素 E/VE、 乳化剂 Montanov 68 加入油相化料锅中， 搅拌加热到 85°C; 2、 将原料甘油、 乳化剂 HR— 透明脂酸 HA、 杰琼一 II和去离子水加至乳化锅中加热搅拌 至 87-89°C; 3、 启动真空泵将乳化锅抽真空到 0.06Mpa， 停止真空， 将油相抽进乳化锅内 高速均质 10分钟， 搅拌转速 70转， 高速均质后停止； 低速均质 10分钟， 停止均质， 继续 搅拌 10分钟， 转速为 60转； 4、 通水慢速冷却， 温度 60°C时加入原料增稠剂 SepiGEL305 和杀螨止痒剂 SM- 650均质 2分钟后真空脱气； 5、 当 45°C时加入原料去离子水和人溶菌酶 溶液， 搅拌至 40°C时停机出料，（在符合 GMP要求的制药工厂， 按制药规程完成） 制成护 肤膏。  Process: 1. Add raw material dimethyl silicone oil 200cst, 18 alcohol, octyl carbonic acid glyceride/GTCC, isooctyl palmitate/GD-5103, vitamin E/VE, emulsifier Montanov 68 into the oil phase chemical pot. , stir and heat to 85 ° C; 2, the raw material glycerin, emulsifier HR - hyaluronic acid HA, Jieqiong II and deionized water was added to the emulsifier pot and heated to 87-89 ° C; 3, start the vacuum pump will The emulsifier is vacuumed to 0.06Mpa, the vacuum is stopped, the oil phase is pumped into the emulsifier pot for high-speed homogenization for 10 minutes, the stirring speed is 70 rpm, and the high-speed homogenization is stopped. The low-speed homogenization is 10 minutes, the homogenization is stopped, and the stirring is continued for 10 minutes. , the speed is 60 rpm; 4, slow cooling through water, the temperature thickener SepiGEL305 and the anti-itch agent SM- 650 homogenized after 2 minutes of vacuum degassing at 60 ° C; 5, when 45 ° C Add the raw material deionized water and human lysozyme solution, stop the output when stirring to 40 °C, (in the GMP-compliant pharmaceutical factory, according to the pharmaceutical regulations) to make a skin cream.
实施例 3: Example 3:
按照实施例 2所述方法制备护肤膏， 配方 （重量） 如下所述：  A skin cream was prepared as described in Example 2, and the formulation (weight) was as follows:
(1) 重组人溶菌酶溶液 100万 U/mL/g (2) 二甲基硅油 200cst 2.0 (1) Recombinant human lysozyme solution 1 million U/mL/g (2) Dimethicone 200cst 2.0
(3) 18醇 5.0  (3) 18 alcohol 5.0
(4) 辛碳癸酸甘油酯 /GTCC 2  (4) Cyanuric acid glyceride / GTCC 2
(5) 棕榈酸异辛酯 /GD— 5103 4.0  (5) Isooctyl palmitate /GD-5103 4.0
(6) 维生素 E/VE 0.3  (6) Vitamin E/VE 0.3
(7) 乳化剂 Montanov 68 1.0  (7) Emulsifier Montanov 68 1.0
(8) 甘油 9  (8) Glycerin 9
(9) 乳化剂 1.0  (9) Emulsifier 1.0
(10) 透明脂酸 HA 0.01  (10) Transparent fatty acid HA 0.01
(11) 增稠剂 SepiGEL305 0.1  (11) Thickener SepiGEL305 0.1
(12)杰玛一 II 1.0  (12) Gemma II II 1.0
(13) 香精 0.10  (13) Fragrance 0.10
(14)去离子水 73.  (14) Deionized water 73.
(15) 杀螨止痒剂 SM- 650 0.5  (15) Killing and anti-itching agent SM- 650 0.5
实施例 4: Example 4:
按照实施例 1所述方法制备人溶菌酶， 按下述配方及工艺制备成治疗痤疮面霜: 配方： （重量)  The human lysozyme was prepared according to the method described in Example 1, and prepared into a therapeutic acne cream according to the following formula and process: Formula: (weight)
(1) 重组人溶菌酶溶液 30万 U/mL/g  (1) Recombinant human lysozyme solution 300,000 U/mL/g
(2) 二甲基硅油 200cst 2.0  (2) Dimethyl silicone oil 200cst 2.0
(3) 18醇 3.0  (3) 18 alcohols 3.0
(4) 辛碳癸酸甘油酯 /GTCC 3  (4) Cyanuric acid glyceride / GTCC 3
(5) 棕榈酸异辛酯 /GD— 5103 4.0  (5) Isooctyl palmitate /GD-5103 4.0
(6) 维生素 EVE 0.2  (6) Vitamin EVE 0.2
(7) 乳化剂 Montanov 68 1.0 (8) 甘油 8 (7) Emulsifier Montanov 68 1.0 (8) Glycerin 8
(9) 乳化剂 HR—S, 1.0  (9) Emulsifier HR-S, 1.0
(10) 透明脂酸 HA 0.01  (10) Transparent fatty acid HA 0.01
(11) 增稠剂 SepiGEL305 0.1  (11) Thickener SepiGEL305 0.1
(12) 杰琼一 II 1.0  (12) Jie Qiongyi II 1.0
(13) 香精 0.1  (13) Fragrance 0.1
(14)去离子水 76.0  (14) Deionized water 76.0
(15) 杀螨止痒剂 SM- 650 0.5  (15) Killing and anti-itching agent SM- 650 0.5
工艺： 1、将原料二甲基硅油 200cst、 18醇、辛碳癸酸甘油酯 /GTCC、棕榈酸异辛酯 /GD 一 5103、 维生素 E/VE、 乳化剂 Montanov 68 加入油相化料锅中， 搅拌加热到 85°C; 2、 将原料甘油、 乳化剂 HR— Si、 透明脂酸 HA、 杰琼一 Π和去离子水加至乳化锅中加热搅拌 至 87-89Ό; 3、 启动真空泵将乳化锅抽真空到 0.06Mpa， 停止真空， 将油相抽进乳化锅内 高速均质 10分钟， 搅拌转速 70转， 高速均质后停止； 低速均质 10分钟， 停止均质， 继续 搅拌 10分钟， 转速为 60转； 4、 通水慢速冷却， 温度 60°C时加入原料增稠剂 SepiGEL305 和杀螨止痒剂 SM- 650均质 2分钟后真空脱气； 5、 当 45°C时加入原料去离子水和人溶菌酶 溶液， 搅拌至 40°C时停机出料，（在符合 GMP要求的制药工厂， 按制药规程完成） 制成面 霜。  Process: 1. Add raw material dimethyl silicone oil 200cst, 18 alcohol, octyl carbonic acid glyceride/GTCC, isooctyl palmitate/GD-5103, vitamin E/VE, emulsifier Montanov 68 into the oil phase chemical pot. , stir and heat to 85 ° C; 2, the raw material glycerin, emulsifier HR - Si, hyaluronic acid HA, Jieqiong and deionized water were added to the emulsifier pot and heated to 87-89 Ό; 3, start the vacuum pump will The emulsifier is vacuumed to 0.06Mpa, the vacuum is stopped, the oil phase is pumped into the emulsifier pot for high-speed homogenization for 10 minutes, the stirring speed is 70 rpm, and the high-speed homogenization is stopped. The low-speed homogenization is 10 minutes, the homogenization is stopped, and the stirring is continued for 10 minutes. , the speed is 60 rpm; 4, slow cooling through water, the temperature thickener SepiGEL305 and the anti-itch agent SM- 650 homogenized after 2 minutes of vacuum degassing at 60 ° C; 5, when 45 ° C Add the raw material deionized water and human lysozyme solution, stop the output when stirring to 40 ° C, (in the GMP-compliant pharmaceutical factory, according to the pharmaceutical regulations) to make a face cream.
实施例 5: Example 5
根据实施例 4所述方法制成面霜， 具体配方 （重量） 如下：  The cream was made according to the method described in Example 4, and the specific formula (weight) was as follows:
(1) 重组人溶菌酶溶液 300万 U/mL/g  (1) Recombinant human lysozyme solution 3 million U/mL/g
(2) 二甲基硅油 200cst 2.0  (2) Dimethyl silicone oil 200cst 2.0
(3) 18醇 3.0  (3) 18 alcohols 3.0
(4) 辛碳癸酸甘油酯 /GTCC 2  (4) Cyanuric acid glyceride / GTCC 2
(5) 棕榈酸异辛酯 /GD— 5103 4.0 (6) 维生素 E/VE 0.2 (5) Isooctyl palmitate / GD — 5103 4.0 (6) Vitamin E/VE 0.2
(7) 乳化剂 Montanov 68 1.0  (7) Emulsifier Montanov 68 1.0
(8) 甘油  (8) Glycerin
(9) 乳化剂 HR—S! 1.0  (9) Emulsifier HR-S! 1.0
(10) 透明脂酸 HA 0.01  (10) Transparent fatty acid HA 0.01
(11) 增稠剂 SepiGEL305 0.1  (11) Thickener SepiGEL305 0.1
(12) 杰琼一 II 1.0  (12) Jie Qiongyi II 1.0
(13) 香精 0.1  (13) Fragrance 0.1
(14)去离子水 75.0  (14) Deionized water 75.0
(15) 杀螨止痒剂 SM- 650 0.5  (15) Killing and anti-itching agent SM- 650 0.5
实施例 6: Example 6:
按照实施例 1所述方法制备人溶菌酶， 然后按照下述配方（重量）制成治疗痤疮乳液: Human lysozyme was prepared as described in Example 1, and then treated to prepare acne emulsion according to the following formulation (weight):
(1) 重组人溶菌酶溶液 200万 U/mL/g (1) Recombinant human lysozyme solution 2 million U/mL/g
(2) 二甲基硅油 200cst 2.  (2) Dimethyl silicone oil 200cst 2.
(3) 18醇 3.  (3) 18 alcohol 3.
(4) 辛碳癸酸甘油酯 /GTCC 2  (4) Cyanuric acid glyceride / GTCC 2
(5) 棕榈酸异辛酯 /GD— 5103 4.0  (5) Isooctyl palmitate /GD-5103 4.0
(6) 维生素 E/VE 0.3  (6) Vitamin E/VE 0.3
(7) 乳化剂 Montanov 68 1  (7) Emulsifier Montanov 68 1
(8) 甘油 5  (8) Glycerin 5
(9) 乳化剂 HR— S】 1.0  (9) Emulsifier HR- S] 1.0
(10) 透明脂酸 HA 0.01  (10) Transparent fatty acid HA 0.01
(11) 杰琼一 II 1.0  (11) Jie Qiongyi II 1.0
(12) 香精 0.1 (13)去离子水 76.0 (12) Fragrance 0.1 (13) Deionized water 76.0
(14) 杀螨止痒剂 SM- 650 0.5  (14) Killing and anti-itching agent SM- 650 0.5
工艺： 1、将原料二甲基硅油 200cst、 18醇、辛碳癸酸甘油酯 /GTCC、棕榈酸异辛酯 /GD 一 5103、 维生素 E/VE、 乳化剂 Montanov 68 加入油相化料锅中， 搅拌加热到 85°C; 2、 将原料甘油、 乳化剂 HR— S,、 透明脂酸 HA、 杰琼一 II和去离子水加至乳化锅中加热搅拌 至 87-89'C; 3、 启动真空泵将乳化锅抽真空到 0.06Mpa， 停止真空， 将油相抽进乳化锅内 高速均质 10分钟， 搅拌转速 70转， 高速均质后停止； 低速均质 10分钟， 停止均质， 继续 搅拌 10分钟， 转速为 60转； 4、 通水慢速冷却， 温度 60°C时加入杀螨止痒剂 SM-650均质 2分钟后真空脱气； 5、 当 45°C时加入原料去离子水和人溶菌酶溶液， 搅拌至 40°C时停机 出料,（在符合 GMP要求的制药工厂， 按制药规程完成） 制成乳液。  Process: 1. Add raw material dimethyl silicone oil 200cst, 18 alcohol, octyl carbonic acid glyceride/GTCC, isooctyl palmitate/GD-5103, vitamin E/VE, emulsifier Montanov 68 into the oil phase chemical pot. , stirring and heating to 85 ° C; 2, the raw material glycerin, emulsifier HR - S, hyaluronic acid HA, Jieqiong II and deionized water was added to the emulsifier pot and heated to 87-89 'C; Start the vacuum pump and vacuum the emulsifier to 0.06Mpa, stop the vacuum, pump the oil phase into the emulsifier pot for 10 minutes at high speed, stir at 70 rpm, stop after high speed homogenization; homogenize at low speed for 10 minutes, stop homogenization, continue Stir for 10 minutes, the speed is 60 rpm; 4, slow cooling through water, temperature 60 ° C, add anti-itch and anti-itch agent SM-650 homogenization after 2 minutes, vacuum degassing; 5, when 45 ° C, add raw materials Ionic water and human lysozyme solution, stop the output when stirring to 40 ° C, (in the pharmaceutical factory in accordance with GMP requirements, according to the pharmaceutical regulations) to make the emulsion.
实施例 7: Example 7
按照实施例 6所述方法制备乳液， 具体配方 （重量） 如下所述：  The emulsion was prepared as described in Example 6, and the specific formulation (weight) was as follows:
(1) 重组人溶菌酶溶液 80万 U/mL/g  (1) Recombinant human lysozyme solution 800,000 U/mL/g
(2) 二甲基硅油 200cst 2.0  (2) Dimethyl silicone oil 200cst 2.0
(3) 18醇 3.  (3) 18 alcohol 3.
(4) 辛碳癸酸甘油酯 /GTCC 2  (4) Cyanuric acid glyceride / GTCC 2
(5) 棕榈酸异辛酯 /GD— 5103 4.0  (5) Isooctyl palmitate /GD-5103 4.0
(6) 维生素 E/VE 0.3  (6) Vitamin E/VE 0.3
(7) 乳化剂 Montanov 68 1.0  (7) Emulsifier Montanov 68 1.0
(8) 甘油 5  (8) Glycerin 5
(9) 乳化剂 HR—St 1.0  (9) Emulsifier HR-St 1.0
(10) 透明脂酸 HA 0.01  (10) Transparent fatty acid HA 0.01
(11) 杰琼—II 1.0  (11) Jieqiong-II 1.0
(12) 香精 0.10 (13)去离子水 76.0 (12) Fragrance 0.10 (13) Deionized water 76.0
( 14) 杀螨止痒剂 SM- 650 Ο.ί  ( 14) Killing and anti-itching agent SM- 650 Ο.ί
实施例 8: Example 8
按照实施例 1所述方法生产人溶菌酶， 然后按照下述配方 （重量） 制成治疗痤疮的面 膜- Human lysozyme was produced according to the method described in Example 1, and then a mask for treating acne was prepared according to the following formula (weight) -
( 1 ) 重组人溶菌酶溶液 6万 U/mL/g (1) Recombinant human lysozyme solution 60,000 U/mL/g
(2) 卡波树脂 CP941 0.4  (2) Kappa resin CP941 0.4
(3) 羧甲基纤维素 CMC 0.5  (3) Carboxymethylcellulose CMC 0.5
(4) 聚乙烯醇 1788 (PVA) 8  (4) Polyvinyl alcohol 1788 (PVA) 8
( 5 ) 甘油 7  ( 5 ) Glycerin 7
(6) 吐温一 80 (T-80) 1.0  (6) Tween 80 (T-80) 1.0
(7) 氮酮 0.6  (7) Azones 0.6
( 8) 乙醇 6  (8) Ethanol 6
(9) 香精 0.3  (9) Fragrance 0.3
( 10) 水 73  (10) Water 73
( 11 ) 杰玛一 II 0. 2  (11) Gemma II II 0. 2
( 12) 杀螨止痒剂 SM- 650纯度为 99%， 0.6  ( 12) Killing and anti-itching agent SM- 650 is 99% pure, 0.6
工艺流程为： 成膜材料浆液配制一加入配方药物一着色剂等一脱泡一涂膜一干燥一脱 膜一含量测定一成品包装。 具体工艺为- The process flow is as follows: a film-forming material slurry is prepared by adding a formula drug, a coloring agent, etc., a defoaming film, a drying film, a film, a content, and a finished product package. The specific process is -
1、 首先将原料卡波树脂 941和羧甲基纤维素 CMC均匀分散于甘油中， 在搅拌情况下 将离子水加入充分混均匀， 此过程应在生产前 4小时备好。 之后搅拌下加入原料聚乙烯醇 1788。 1. Firstly, the raw material Kappa resin 941 and carboxymethyl cellulose CMC are uniformly dispersed in glycerin, and the ionized water is thoroughly mixed and mixed under stirring, and the process should be prepared 4 hours before the production. The raw material polyvinyl alcohol 1788 was then added with stirring.
2、将搅拌均匀的 1加入乳化锅内， 然后加入吐温一 80、氮酮和水溶性硅油搅拌加热到 75 °C , 启动均质， 高速均质 10分钟， 然后停止高速均质， 低速均质 15分钟后停止， 再继 续搅拌 10分钟， 搅拌转速为 60转 /分。 2. Add the evenly stirred 1 into the emulsification pot, then add Tween 80, azone and water-soluble silicone oil and stir to heat to 75 °C, start homogenization, homogenize at high speed for 10 minutes, then stop high-speed homogenization, low speed Stop after 15 minutes, continue Stirring was continued for 10 minutes, and the stirring speed was 60 rpm.
3、 通过冷却水使其慢速冷却， 当温度达到 50°C加入乙醇和香精和杀螨止痒剂 SM- 650 然后继续搅拌到冷却， 并在温度低于 45°C时加入人溶菌酶和杰琼后均质 2分钟， 之后进行 真空脱气。 搅拌到物料 40Ό停机出料。 制备成治疗痤疮的面膜成品。 （此膜剂型要在符合 GMP要求的制药工厂， 按制药规程完成）。  3, through the cooling water to slow cooling, when the temperature reaches 50 ° C, add ethanol and flavor and killing anti-itch agent SM- 650 and then continue to stir to cool, and add human lysozyme and when the temperature is lower than 45 ° C After Jieqiong, it was homogenized for 2 minutes, and then vacuum degassed. Stir to the material 40 Ό stop the discharge. Prepared as a finished mask for treating acne. (This film type should be completed in accordance with GMP regulations in pharmaceutical factories that meet GMP requirements).
实施例 9: Example 9
按照实施例 8所述方法制备治疗痤疮的面膜， 具体配方 （重量） 如下：  A mask for treating acne was prepared according to the method described in Example 8, and the specific formulation (weight) was as follows:
(1) 重组人溶菌酶溶液 182万 U/mL/g  (1) Recombinant human lysozyme solution 1.82 million U/mL/g
(2) 卡波树脂 CP941 0.3  (2) Kappa resin CP941 0.3
(3) 羧甲基纤维素 CMC 0.4  (3) Carboxymethylcellulose CMC 0.4
(4) 聚乙烯醇 1788 (PVA) 9  (4) Polyvinyl alcohol 1788 (PVA) 9
(5) 甘油 6  (5) Glycerin 6
(6) 吐温一 80 (T— 80) 1.2  (6) Tween 80 (T-80) 1.2
(7) 氮酮 0.8  (7) Azones 0.8
(8) 乙醇 6  (8) Ethanol 6
(9) 香精 0.2  (9) Fragrance 0.2
(10) 水 75  (10) Water 75
(11) 杰玛— Π 0.2  (11) Gemma - Π 0.2
(12) 杀螨止痒剂 SM- 650纯度为 99% 0.8。  (12) Killing and anti-itching agent SM-650 has a purity of 99% 0.8.

Claims

权利 要求 Rights request
1、 人溶菌酶在制备治疗粉刺或痤疮的化妆品中的应用。 1. The application of human lysozyme in the preparation of cosmetics for treating acne or acne.
2、人溶菌酶在制备治疗由金黄色葡萄球菌、表葡菌、丙酸杆菌或螨虫引起的痤疮的化 妆品中的应用。  2. The use of human lysozyme in the preparation of a cosmetic for treating acne caused by Staphylococcus aureus, epiphylococcus, Propionibacterium or aphids.
3、 根据权利要求 1或 2所述人溶菌酶在制备治疗痤疮的化妆品中的应用， 其特征是： 人溶菌酶为基因工程表达的重组人溶菌酶或基因.工程表达人溶菌酶的氨基端带有 （谷氨酸 一丙氨酸） 2或 （谷氨酸一丙氨酸） 3修饰的人溶菌酶或基因工程表达或化学合成突变体重 组人溶菌酶。 3. The use of human lysozyme according to claim 1 or 2 for the preparation of a cosmetic for treating acne, characterized in that: human lysozyme is a recombinant human lysozyme or gene expressed by genetic engineering. The amino terminal of engineering lysozyme is expressed. Human lysozyme or genetically engineered or chemically synthesized mutant recombinant human lysozyme with (glutamate-alanine) 2 or (glutamate-alanine) 3 modification.
4、 根据权利要求 1或 2所述人溶菌酶在制备治疗痤疮的化妆品中的应用， 其特征是： 所述化妆品中含有人溶菌酶 300U〜300万 U/mL/g。  The use of human lysozyme according to claim 1 or 2 for the preparation of a cosmetic for treating acne, characterized in that: the cosmetic contains human lysozyme 300 U to 3 million U/mL/g.
5、 根据权利要求 1或 2所述人溶菌酶在制备治疗痤疮的化妆品中的应用， 其特征是： 化妆品为洗面奶、 化妆水、 护肤膏、 面霜、 乳液或面膜。  5. The use of human lysozyme according to claim 1 or 2 for the preparation of a cosmetic for treating acne, characterized in that the cosmetic is a facial cleanser, a lotion, a skin cream, a face cream, an emulsion or a mask.
6、根据权利要求 5所述人溶菌酶在制备治疗痤疮的化妆品中的应用， 其特征是： 将纯 度 95%〜99%的基因重组人溶菌酶制得 1500U〜30 万 U/ml/g， 磷酸盐缓冲液 10〜 20mM(pH6. 0〜7. 5)75〜85%、 5〜 25%丙二醇或丙三醇、 5〜15% 2—吡咯烷酮一 5—羧酸钠、 万分之 1〜5吐温 80, 1〜5%水溶性氮酮、 尼泊金 0. 01〜0. 05%、 山梨酸钾 0. 01〜0. 1%, 在 常温下混合均质， 制成化妆水。  6. The use of human lysozyme according to claim 5 for the preparation of a cosmetic for treating acne, characterized in that: 1500 U to 300,000 U/ml/g of recombinant human lysozyme having a purity of 95% to 99% is obtained. Phosphate buffer 10~20mM (pH6. 0~7. 5) 75~85%, 5~ 25% propylene glycol or glycerol, 5~15% 2-pyrrolidone 5-carboxylic acid sodium, 1~5~5 01〜0. 05%, potassium sorbate 0. 01~0. 1%, mixed at room temperature, and made into a lotion. Tween 80, 1~5% water-soluble azone, 0. 01~0. 05%, potassium sorbate 0. 01~0.
7、根据权利要求 5所述人溶菌酶在制备治疗痤疮的化妆品中的应用， 其特征是： 所述 化妆品为护肤膏， 其配方重量份比为：  7. The use of human lysozyme according to claim 5 for the preparation of a cosmetic for treating acne, characterized in that: the cosmetic is a skin care cream, and the weight ratio of the formulation is:
( 1 ) 重组人溶菌酶溶液 300U—300万 U/mL/g  (1) Recombinant human lysozyme solution 300U-3 million U/mL/g
(2) 二甲基硅油 200cst 2.0-4.0  (2) Dimethyl silicone oil 200cst 2.0-4.0
(3 ) 18醇 3.0-6.0  (3) 18 alcohol 3.0-6.0
(4) 辛碳癸酸甘油酯 /GTCC 2-5.0 (5) 棕榈酸异辛酯 /GD— 5103 4.0-7.0 (4) Cyanuric acid glyceride / GTCC 2-5.0 (5) Isooctyl palmitate/GD-5103 4.0-7.0
(6) 维生素 E/VE 0.3-0.7  (6) Vitamin E/VE 0.3-0.7
(7) 乳化剂 Montanov 68 1.0-3.0  (7) Emulsifier Montanov 68 1.0-3.0
(8) 甘油 5-15  (8) Glycerin 5-15
(9) 乳化剂 HR—S, 1.0-3.0  (9) Emulsifier HR-S, 1.0-3.0
(10) 透明脂酸 HA 0.01-0.04  (10) Transparent fatty acid HA 0.01-0.04
(11) 增稠剂 SepiGEL305 1.0-3.0  (11) Thickener SepiGEL305 1.0-3.0
(12) 杰玛— Π 1.0-2.0  (12) Gemma - Π 1.0-2.0
(13) 香精 0.10-0.20  (13) Fragrance 0.10-0.20
(14)去离子水 70.0-75.0  (14) Deionized water 70.0-75.0
(15) 杀螨止痒剂 SM- 650 0.5〜1。  (15) Killing and antipruritic agent SM-650 0.5~1.
8、根据权利要求 5所述人溶菌酶在制备治疗痤疮的化妆品中的应用， 其特征是： 所述 品为面霜， 配方重量份比为:  8. The use of human lysozyme according to claim 5 for the preparation of a cosmetic for treating acne, characterized in that: the product is a face cream, and the weight ratio of the formula is:
(1) 重组人溶菌酶溶液 300U—300万 U/mL/g  (1) Recombinant human lysozyme solution 300U-3 million U/mL/g
(2) 二甲基硅油 200cst 2.0-4.0  (2) Dimethyl silicone oil 200cst 2.0-4.0
(3) 18醇 3.0-6.0  (3) 18 alcohol 3.0-6.0
(4) 辛碳癸酸甘油酯 /GTCC 2-5.0  (4) Cyanuric acid glyceride / GTCC 2-5.0
(5) 棕榈酸异辛酯 /GD— 5103 4.0-7.0  (5) Isooctyl palmitate /GD-5103 4.0-7.0
(6) 维生素 E/VE 0.5-1.2  (6) Vitamin E/VE 0.5-1.2
(7) 乳化剂 Montanov 68 1.0-3.0  (7) Emulsifier Montanov 68 1.0-3.0
(8) 甘油 5-8  (8) Glycerin 5-8
(9) 乳化剂 HR—Sj 1.0-3.0  (9) Emulsifier HR-Sj 1.0-3.0
(10) 透明脂酸 HA 0.01-0.04  (10) Transparent fatty acid HA 0.01-0.04
(11) 增稠剂 SepiGEL305 0.8-1. (12) 杰琼— II 1.0-2.0 (11) Thickener SepiGEL305 0.8-1. (12) Jieqiong - II 1.0-2.0
(13) 香精 0.10-0.20  (13) Fragrance 0.10-0.20
(14)去离子水 70.0-75,0  (14) Deionized water 70.0-75,0
(15) 杀螨止痒剂 SM-650 0.5—1 。  (15) Killing and antipruritic agent SM-650 0.5-1.
9、根据权利要求 5所述人溶菌酶在制备治疗痤疮的化妆品中的应用， 其特征是： 护肤 乳液的配方重量份比为：  9. The use of human lysozyme according to claim 5 for the preparation of a cosmetic for treating acne, characterized in that: the formula weight ratio of the skin care lotion is:
(1) 重组人溶菌酶溶液 300U〜300万 U/mL/g  (1) Recombinant human lysozyme solution 300U~3 million U/mL/g
(2) 二甲基硅油 200cst 2.0-5.0  (2) Dimethyl silicone oil 200cst 2.0-5.0
(3) 18醇 3.-5.0  (3) 18 alcohol 3.-5.0
(4) 辛碳癸酸甘油酯 /GTCC 2-5.0  (4) Cyanuric acid glyceride / GTCC 2-5.0
(5) 棕榈酸异辛酯 /GD— 5103 4.0-7.0  (5) Isooctyl palmitate /GD-5103 4.0-7.0
(6) 维生素 E/VE 0.3-0.7  (6) Vitamin E/VE 0.3-0.7
(7) 乳化剂 Montanov 68 1.0-3.0  (7) Emulsifier Montanov 68 1.0-3.0
(8) 甘油 5-8  (8) Glycerin 5-8
(9) 乳化剂 HR—Si 1.0-4.0  (9) Emulsifier HR-Si 1.0-4.0
(10) 透明脂酸 HA 0.01-0.04  (10) Transparent fatty acid HA 0.01-0.04
(11) 杰琼一 II 1.0-2.0  (11) Jie Qiongyi II 1.0-2.0
(12) 香精 0.10-0.20  (12) Fragrance 0.10-0.20
(13)去离子水 70.0-76.0  (13) Deionized water 70.0-76.0
(14) 杀螨止痒剂 SM- 650  (14) Killing and anti-itching agent SM- 650
10、 根据权利要求 5所述人溶菌酶在制备治疗痤疮的化妆品中的应用， 其特征是： 面膜的 配方重量份比为:  10. The use of human lysozyme according to claim 5 for the preparation of a cosmetic for treating acne, characterized in that: the formula weight ratio of the mask is:
(1) 重组人溶菌酶溶液 300U〜300万 U/mL/g  (1) Recombinant human lysozyme solution 300U~3 million U/mL/g
(2) 卡波树脂 CP941 0.2-0. (3) 羧甲基纤维素 CMC 0.3-0.6(2) Kappa resin CP941 0.2-0. (3) Carboxymethyl cellulose CMC 0.3-0.6
(4) 聚乙烯醇 1788 (PVA) 7-10(4) Polyvinyl alcohol 1788 (PVA) 7-10
(5) 甘油 6-8(5) Glycerin 6-8
(6) 吐温 _80 (T— 80) 0.5-1.5(6) Tween _80 (T-80) 0.5-1.5
(7) 氮酮 0.3-1.0(7) Azones 0.3-1.0
(8) 乙醇 5-10(8) Ethanol 5-10
(9) 香精 0.1-0.5(9) Fragrance 0.1-0.5
(10) 水 67.6-80.45(10) Water 67.6-80.45
(11) 杰玛一 II 0.15-0.3(11) Gemma II II 0.15-0.3
(12) 杀螨止痒剂 SM- 650 0.5—1 。 (12) Killing and antipruritic agent SM- 650 0.5-1.
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CN1439427A (en) * 2003-01-09 2003-09-03 长春奇龙生物技术研究所 Use of gene recombination human lysozyme against drug resistant bacteria in preparation of medicine
CN1557485A (en) * 2004-01-30 2004-12-29 大连帝恩生物工程有限公司 Application of gene recombined human lysozyme in eliminating pathogenic microorganism infection
CN1583169A (en) * 2004-06-10 2005-02-23 安米 Use of human lysozyme in preparation of medicine for hemorrhoid
CN1593652A (en) * 2004-06-21 2005-03-16 张华� Usage of human lysozyme in preparation of dermics
CN1634574A (en) * 2004-11-12 2005-07-06 张华� Use of human lysozyme for preparing medicine to treat intertrigo
CN1634573A (en) * 2004-11-12 2005-07-06 张华� Use of human lysozyme for preparing medicine to treat prickly heat
CN1679503A (en) * 2005-01-07 2005-10-12 张华� Human lysozyme prickly-heat powder and its preparation

Cited By (5)

* Cited by examiner, † Cited by third party
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EP1774975A1 (en) * 2004-06-10 2007-04-18 Mi An Use of human lysozyme in preparation of medicine for treating acne
EP1774975A4 (en) * 2004-06-10 2008-09-03 Mi An Use of human lysozyme in preparation of medicine for treating acne
CN109799335A (en) * 2019-01-31 2019-05-24 陕西慧康生物科技有限责任公司 The detection method of Pichia pastoris host protein residual quantity in recombinant human lysozyme
CN114699336A (en) * 2022-05-23 2022-07-05 浙江青康生物科技有限公司 Mask containing lysozyme
CN114699336B (en) * 2022-05-23 2023-11-28 浙江青康生物科技有限公司 Facial mask containing lysozyme

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