CN108815180A - 一种pm2.5致肺损伤拮抗剂及其制备方法 - Google Patents
一种pm2.5致肺损伤拮抗剂及其制备方法 Download PDFInfo
- Publication number
- CN108815180A CN108815180A CN201810499088.4A CN201810499088A CN108815180A CN 108815180 A CN108815180 A CN 108815180A CN 201810499088 A CN201810499088 A CN 201810499088A CN 108815180 A CN108815180 A CN 108815180A
- Authority
- CN
- China
- Prior art keywords
- lungs
- antagonist
- sfmp
- preparation
- added
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 210000004072 lung Anatomy 0.000 title claims abstract description 18
- 239000005557 antagonist Substances 0.000 title claims abstract description 17
- 230000006378 damage Effects 0.000 title claims abstract description 16
- 238000002360 preparation method Methods 0.000 title claims abstract description 11
- 208000027418 Wounds and injury Diseases 0.000 title claims description 13
- 208000014674 injury Diseases 0.000 title claims description 13
- 239000005022 packaging material Substances 0.000 claims abstract description 64
- 229920001282 polysaccharide Polymers 0.000 claims abstract description 37
- 239000005017 polysaccharide Substances 0.000 claims abstract description 37
- 150000004676 glycans Chemical class 0.000 claims abstract description 36
- 240000002769 Morchella esculenta Species 0.000 claims abstract description 15
- 235000002779 Morchella esculenta Nutrition 0.000 claims abstract description 15
- WQZGKKKJIJFFOK-QTVWNMPRSA-N D-mannopyranose Chemical compound OC[C@H]1OC(O)[C@@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-QTVWNMPRSA-N 0.000 claims abstract description 4
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims abstract description 4
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 claims abstract description 4
- 239000008103 glucose Substances 0.000 claims abstract description 4
- 238000006467 substitution reaction Methods 0.000 claims abstract description 4
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 15
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 8
- 241000723418 Carya Species 0.000 claims description 7
- ZHNUHDYFZUAESO-UHFFFAOYSA-N Formamide Chemical compound NC=O ZHNUHDYFZUAESO-UHFFFAOYSA-N 0.000 claims description 6
- 238000000502 dialysis Methods 0.000 claims description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 6
- 235000019441 ethanol Nutrition 0.000 claims description 5
- 230000001376 precipitating effect Effects 0.000 claims description 5
- 238000006243 chemical reaction Methods 0.000 claims description 4
- 239000000203 mixture Substances 0.000 claims description 3
- 238000004108 freeze drying Methods 0.000 claims description 2
- 150000002772 monosaccharides Chemical class 0.000 claims description 2
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 claims 1
- 239000000654 additive Substances 0.000 claims 1
- 230000000996 additive effect Effects 0.000 claims 1
- 239000008101 lactose Substances 0.000 claims 1
- 230000000694 effects Effects 0.000 abstract description 14
- 230000008485 antagonism Effects 0.000 abstract description 5
- 230000004048 modification Effects 0.000 abstract description 5
- 238000012986 modification Methods 0.000 abstract description 5
- 230000036541 health Effects 0.000 abstract description 4
- 230000001681 protective effect Effects 0.000 abstract description 3
- 210000001132 alveolar macrophage Anatomy 0.000 abstract description 2
- 239000003814 drug Substances 0.000 abstract description 2
- 230000002685 pulmonary effect Effects 0.000 abstract 1
- 210000004027 cell Anatomy 0.000 description 32
- 239000000243 solution Substances 0.000 description 15
- 230000006907 apoptotic process Effects 0.000 description 13
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 10
- 108060008682 Tumor Necrosis Factor Proteins 0.000 description 10
- 102000000852 Tumor Necrosis Factor-alpha Human genes 0.000 description 10
- MZOFCQQQCNRIBI-VMXHOPILSA-N (3s)-4-[[(2s)-1-[[(2s)-1-[[(1s)-1-carboxy-2-hydroxyethyl]amino]-4-methyl-1-oxopentan-2-yl]amino]-5-(diaminomethylideneamino)-1-oxopentan-2-yl]amino]-3-[[2-[[(2s)-2,6-diaminohexanoyl]amino]acetyl]amino]-4-oxobutanoic acid Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](CC(O)=O)NC(=O)CNC(=O)[C@@H](N)CCCCN MZOFCQQQCNRIBI-VMXHOPILSA-N 0.000 description 9
- 102000003777 Interleukin-1 beta Human genes 0.000 description 9
- 108090000193 Interleukin-1 beta Proteins 0.000 description 9
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 7
- 230000004083 survival effect Effects 0.000 description 7
- 238000001514 detection method Methods 0.000 description 5
- 230000006698 induction Effects 0.000 description 5
- 230000031700 light absorption Effects 0.000 description 5
- 108010010803 Gelatin Proteins 0.000 description 4
- 229920000869 Homopolysaccharide Polymers 0.000 description 4
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 4
- 230000005779 cell damage Effects 0.000 description 4
- 230000001419 dependent effect Effects 0.000 description 4
- 229920000159 gelatin Polymers 0.000 description 4
- 239000008273 gelatin Substances 0.000 description 4
- 235000019322 gelatine Nutrition 0.000 description 4
- 235000011852 gelatine desserts Nutrition 0.000 description 4
- WFDIJRYMOXRFFG-UHFFFAOYSA-N Acetic anhydride Chemical compound CC(=O)OC(C)=O WFDIJRYMOXRFFG-UHFFFAOYSA-N 0.000 description 3
- 108010019160 Pancreatin Proteins 0.000 description 3
- 230000000052 comparative effect Effects 0.000 description 3
- 235000009508 confectionery Nutrition 0.000 description 3
- 230000000254 damaging effect Effects 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 230000014759 maintenance of location Effects 0.000 description 3
- 238000000034 method Methods 0.000 description 3
- 230000020477 pH reduction Effects 0.000 description 3
- 229940055695 pancreatin Drugs 0.000 description 3
- 238000012545 processing Methods 0.000 description 3
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 2
- 229920002307 Dextran Polymers 0.000 description 2
- 241000233866 Fungi Species 0.000 description 2
- 238000004566 IR spectroscopy Methods 0.000 description 2
- 206010061218 Inflammation Diseases 0.000 description 2
- 230000021736 acetylation Effects 0.000 description 2
- 238000006640 acetylation reaction Methods 0.000 description 2
- RJURFGZVJUQBHK-UHFFFAOYSA-N actinomycin D Natural products CC1OC(=O)C(C(C)C)N(C)C(=O)CN(C)C(=O)C2CCCN2C(=O)C(C(C)C)NC(=O)C1NC(=O)C1=C(N)C(=O)C(C)=C2OC(C(C)=CC=C3C(=O)NC4C(=O)NC(C(N5CCCC5C(=O)N(C)CC(=O)N(C)C(C(C)C)C(=O)OC4C)=O)C(C)C)=C3N=C21 RJURFGZVJUQBHK-UHFFFAOYSA-N 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 230000003078 antioxidant effect Effects 0.000 description 2
- TZCXTZWJZNENPQ-UHFFFAOYSA-L barium sulfate Chemical compound [Ba+2].[O-]S([O-])(=O)=O TZCXTZWJZNENPQ-UHFFFAOYSA-L 0.000 description 2
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 2
- 239000006285 cell suspension Substances 0.000 description 2
- 230000001413 cellular effect Effects 0.000 description 2
- 238000012512 characterization method Methods 0.000 description 2
- 239000008367 deionised water Substances 0.000 description 2
- 229910021641 deionized water Inorganic materials 0.000 description 2
- 230000029087 digestion Effects 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 238000004090 dissolution Methods 0.000 description 2
- 238000001035 drying Methods 0.000 description 2
- 235000013399 edible fruits Nutrition 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- 238000004128 high performance liquid chromatography Methods 0.000 description 2
- 230000004054 inflammatory process Effects 0.000 description 2
- 230000003834 intracellular effect Effects 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 230000011987 methylation Effects 0.000 description 2
- 238000007069 methylation reaction Methods 0.000 description 2
- 239000012452 mother liquor Substances 0.000 description 2
- IOLCXVTUBQKXJR-UHFFFAOYSA-M potassium bromide Chemical compound [K+].[Br-] IOLCXVTUBQKXJR-UHFFFAOYSA-M 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 230000000638 stimulation Effects 0.000 description 2
- QAOWNCQODCNURD-UHFFFAOYSA-L sulfate group Chemical group S(=O)(=O)([O-])[O-] QAOWNCQODCNURD-UHFFFAOYSA-L 0.000 description 2
- 239000000725 suspension Substances 0.000 description 2
- 238000000108 ultra-filtration Methods 0.000 description 2
- 208000024172 Cardiovascular disease Diseases 0.000 description 1
- 108010092160 Dactinomycin Proteins 0.000 description 1
- 238000002965 ELISA Methods 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 229960000583 acetic acid Drugs 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- RJURFGZVJUQBHK-IIXSONLDSA-N actinomycin D Chemical compound C[C@H]1OC(=O)[C@H](C(C)C)N(C)C(=O)CN(C)C(=O)[C@@H]2CCCN2C(=O)[C@@H](C(C)C)NC(=O)[C@H]1NC(=O)C1=C(N)C(=O)C(C)=C2OC(C(C)=CC=C3C(=O)N[C@@H]4C(=O)N[C@@H](C(N5CCC[C@H]5C(=O)N(C)CC(=O)N(C)[C@@H](C(C)C)C(=O)O[C@@H]4C)=O)C(C)C)=C3N=C21 RJURFGZVJUQBHK-IIXSONLDSA-N 0.000 description 1
- 230000029936 alkylation Effects 0.000 description 1
- 238000005804 alkylation reaction Methods 0.000 description 1
- 230000000259 anti-tumor effect Effects 0.000 description 1
- 239000003963 antioxidant agent Substances 0.000 description 1
- 235000006708 antioxidants Nutrition 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 238000011088 calibration curve Methods 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 125000003636 chemical group Chemical group 0.000 description 1
- 238000001311 chemical methods and process Methods 0.000 description 1
- 238000007385 chemical modification Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000004440 column chromatography Methods 0.000 description 1
- 230000009514 concussion Effects 0.000 description 1
- 229960000640 dactinomycin Drugs 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000004043 dyeing Methods 0.000 description 1
- 239000010419 fine particle Substances 0.000 description 1
- 239000000796 flavoring agent Substances 0.000 description 1
- 235000019634 flavors Nutrition 0.000 description 1
- 239000002803 fossil fuel Substances 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- 230000008014 freezing Effects 0.000 description 1
- 239000012362 glacial acetic acid Substances 0.000 description 1
- 238000000227 grinding Methods 0.000 description 1
- 230000007365 immunoregulation Effects 0.000 description 1
- 230000002757 inflammatory effect Effects 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 210000002540 macrophage Anatomy 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 239000002808 molecular sieve Substances 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 230000035764 nutrition Effects 0.000 description 1
- 238000011017 operating method Methods 0.000 description 1
- 230000004792 oxidative damage Effects 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 239000013618 particulate matter Substances 0.000 description 1
- 239000008188 pellet Substances 0.000 description 1
- 230000026731 phosphorylation Effects 0.000 description 1
- 238000006366 phosphorylation reaction Methods 0.000 description 1
- 239000002574 poison Substances 0.000 description 1
- 231100000614 poison Toxicity 0.000 description 1
- OTYBMLCTZGSZBG-UHFFFAOYSA-L potassium sulfate Chemical compound [K+].[K+].[O-]S([O-])(=O)=O OTYBMLCTZGSZBG-UHFFFAOYSA-L 0.000 description 1
- 229910052939 potassium sulfate Inorganic materials 0.000 description 1
- 235000011151 potassium sulphates Nutrition 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 238000004393 prognosis Methods 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 230000003578 releasing effect Effects 0.000 description 1
- 210000002345 respiratory system Anatomy 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- URGAHOPLAPQHLN-UHFFFAOYSA-N sodium aluminosilicate Chemical compound [Na+].[Al+3].[O-][Si]([O-])=O.[O-][Si]([O-])=O URGAHOPLAPQHLN-UHFFFAOYSA-N 0.000 description 1
- FDRCDNZGSXJAFP-UHFFFAOYSA-M sodium chloroacetate Chemical compound [Na+].[O-]C(=O)CCl FDRCDNZGSXJAFP-UHFFFAOYSA-M 0.000 description 1
- 239000012086 standard solution Substances 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 238000012549 training Methods 0.000 description 1
- YNJBWRMUSHSURL-UHFFFAOYSA-N trichloroacetic acid Chemical compound OC(=O)C(Cl)(Cl)Cl YNJBWRMUSHSURL-UHFFFAOYSA-N 0.000 description 1
- 238000004879 turbidimetry Methods 0.000 description 1
- 238000002604 ultrasonography Methods 0.000 description 1
- 238000003809 water extraction Methods 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
- A61K31/737—Sulfated polysaccharides, e.g. chondroitin sulfate, dermatan sulfate
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/125—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives containing carbohydrate syrups; containing sugars; containing sugar alcohols; containing starch hydrolysates
-
- 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
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Molecular Biology (AREA)
- Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Veterinary Medicine (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Polymers & Plastics (AREA)
- Organic Chemistry (AREA)
- Public Health (AREA)
- Animal Behavior & Ethology (AREA)
- Biochemistry (AREA)
- Dermatology (AREA)
- Food Science & Technology (AREA)
- Epidemiology (AREA)
- Nutrition Science (AREA)
- Mycology (AREA)
- Materials Engineering (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Pulmonology (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Polysaccharides And Polysaccharide Derivatives (AREA)
Abstract
本发明公开了一种PM2.5致肺损伤拮抗剂及其制备方法,该拮抗剂由羊肚菌子实体多糖经硫酸化修饰获得,主要由甘露糖、葡萄糖和半乳糖组成,分子量为9.0kDa,硫酸根取代度为1.36。本发明获得的多糖组分SFMP‑1对于PM2.5损伤的肺泡巨噬细胞具有很好的保护作用,且无明显毒副作用,显示出其具有良好的拮抗PM2.5损伤作用,在医药及保健品等领域具有巨大的应用前景。
Description
技术领域
本发明涉及一种PM2.5致肺损伤拮抗剂及其制备方法,属于生物技术领域。
背景技术
近几年,随着我国经济的迅猛发展以及化石燃料的大面积使用,我们正在面临并遭受着严峻的考验—大气污染。其中包含PM2.5。PM2.5,又称细颗粒物,它是指一种直径小于或等于2.5μm的颗粒物。我国现在的PM2.5水平要比世界卫生组织推荐的正常值高出八倍,被认为是世界上PM2.5水平最高的国家之一。尤其是华北平原,频繁的雾霾天气给人民的生活和出行带来了深重的灾难。由于PM2.5粒子的尺寸,它可以进入到人类的肺部和血液中,给生物系统带来引起严重的影响,甚至会摧毁人类的健康。根据一些风险疾病的评估,雾霾已经成为东亚地区第四大高危风险,且已经导致了中国120万人死亡。流行性病学研究进一步指出长期暴露于室外的雾霾中尤其是PM2.5中还会增加罹患呼吸系统和心血管系统疾病的风险。因此寻找PM2.5拮抗剂具有重要的科学意义和应用价值。
长期以来,食用菌多糖因其独特的免疫调节作用而受到广泛关注。羊肚菌作为一种味道鲜美、营养丰富的珍稀食用菌,其醇提物和粗多糖具有抗肿瘤、护肝、抗氧化等活性。实验室前期从羊肚菌子实体中提取的多糖均一组分FMP-1具有很好的抗氧化活性,而对于拮抗PM2.5致肺损伤的活性还未见报道。此外,研究表明多糖的化学修饰可以通过引入新的化学基团提高自身的生物活性以及产生新的活性,常见的多糖化学修饰方法有多糖的硫酸化、羧甲基化、乙酰化、磷酸化以及烷基化等,通过适当的分子修饰获得高效的多糖基PM2.5拮抗剂也未见报道。
发明内容
本发明的目的在于提供一种PM2.5致肺损伤拮抗剂及其制备方法,无任何毒副作用,且具有良好的抵抗PM2.5致肺泡巨噬细胞损伤作用,在医药及保健品等领域具有巨大的应用前景。
本发明PM2.5致肺损伤拮抗剂,为羊肚菌硫酸化多糖SFMP-1,分子量为9.0kDa,硫酸根取代度为1.36,其单糖组成主要有甘露糖、葡萄糖和半乳糖,摩尔比为1:7.84:1.24。
本发明PM2.5致肺损伤拮抗剂的制备方法,包括如下步骤:
步骤1:取100mg干燥的羊肚菌多糖FMP-1置于洁净干燥的三颈烧瓶,加入甲酰胺5mL,25℃下水浴30min,随后加入200mg SO3-Py,70℃下油浴搅拌反应3h;
步骤2:用NaOH溶液(4mol/L)调步骤1获得的反应液的pH值至中性,透析,所得透析液浓缩,加入四倍体积的95vt%乙醇,4℃下静置12h,离心,收集沉淀,所得沉淀经冷冻干燥得到羊肚菌硫酸化多糖SFMP-1。
步骤2中,所述透析是采用3500Da透析袋,流水透析48h。
步骤2中,所述冷冻干燥的参数为:温度-50℃,气压0.1mbar,时间72h。
本发明PM2.5致肺损伤拮抗剂的表征,包括如下内容:
1、采用硫酸钡比浊法测定硫酸基团含量。配制1mol/L的HCl溶液200mL,称干燥至恒重的硫酸钾108.75mg,用配好的盐酸溶液定容至100mL,配制3%的三氯乙酸溶液250mL,配制BaCl-明胶(1%:0.5%)溶液100mL及0.5%的明胶溶液100mL。按照表4-3的顺序加入各试剂,25℃下静置15min后360nm处测定吸光值Ai。再以0.5%的明胶溶液代替BaCl-明胶(1%:0.5%)溶液重复上述实验,在360nm处测定吸光值Aj。以(Ai-Aj)对硫酸基团含量作标准曲线。配制1mg/mL的样品盐酸溶液,100℃水浴3h,按上述方法测360nm处的吸光值测得Ai样品和Aj样品,测定硫酸化多糖硫酸根含量,按照标准曲线和公式计算出硫酸根取代度。
2、准确配制2mg/mL标准葡聚糖T3,T7,T10,T50,T100,T-500,T-1000标准溶液,按以上条件进行实验。以保留时间t为横坐标,标准葡聚糖分子量的自然对数为纵坐标,绘制相对分子质量标准曲线。将所述的PM2.5致肺损伤拮抗剂的保留时间代入标准曲线方程,求出样品的平均分子量。
3、采用溴化钾压片法对羊肚菌子实体多糖FMP-1的红外光谱结构进行分析。称取适量预先干燥好的光谱纯溴化钾与少量多糖样品混合研磨,压制成薄片,在400~4000cm-1的频率范围内进行红外测定。
本发明PM2.5致肺损伤拮抗剂的活性检测,包括如下内容:
1、利用200μg/mL的PM2.5处理NR8383细胞4h建立起PM2.5损伤细胞模型,通过FMP-1、SFMP-1、CFMP-1和AFMP-1的干预处理,比较相同浓度下(50,100和200μg/mL)不同多糖处理组的细胞存活率、细胞凋亡率发现这四种多糖均可以保护细胞拮抗PM2.5的损伤,其中SFMP-1的效果更加明显,在200μg/mL的浓度下可以将细胞存活率提高至82%;通过流式细胞仪结果的分析得出,经过四种多糖处理的细胞的凋亡率显著下降,且呈现浓度依赖性,尤其是200μg/mL的SFMP-1的凋亡率降低为8.06%,与空白对照组(6.74%)相差不大;
2、通过对细胞炎症因子TNF-α和IL-1β释放量的检测发现,与其他三组多糖比较,SFMP-1降低PM2.5诱导的TNF-α和IL-1β释放效果更加显著,并且呈现明显地浓度依赖性;
3、DCFH-DA染色、流式细胞仪检测的结果显示了伴随着SFMP-1浓度的增加,PM2.5引起的细胞内ROS的产生量也随之降低,在浓度达到200μg/mL时,ROS产生量与空白对照相当。
与现有技术相比,本发明的有益效果体现在:
1、本发明成功得到硫酸化修饰的羊肚菌子实体多糖SFMP-1,步骤简单,效率高。
2、通过对PM2.5损伤的细胞模型的干预发现,SFMP-1可以有效提高损伤细胞模型的生存率;通过细胞凋亡和ELISA实验发现,SFMP-1可以减少PM2.5造成的细胞凋亡,降低PM2.5诱导产生的细胞炎症因子TNF-α和IL-1β水平,并且呈现浓度依赖性,帮助细胞提高抵抗PM2.5损伤能力;此外,细胞在SFMP-1的干预下,PM2.5引起的ROS释放量明显降低,说明SFMP-1可以减弱PM2.5引起的氧化损伤。目前的文献查阅尚未发现天然多糖及其衍生物的相关活性报道,其在拮抗PM2.5致肺损伤方面具有广阔的应用前景。
附图说明
图1是羊肚菌多糖FMP-1和硫酸化修饰的羊肚菌多糖SFMP-1高效液相色谱(HPLC)检测图。从图1中可以看出SFMP为纯品,保留时间为9.78min,根据多糖分子量标准曲线可以算出FMP的分子量为9.0×103Da。
图2是FMP-1和硫酸化多糖SFMP-1红外光谱(FT-IR)检测图。从图2中可以看出红线代表的SFMP-1在1238cm-1和820cm-1处出现了两个新的硫酸根特征吸收峰,说明硫酸化修饰成功。
图3是不同多糖对PM2.5刺激的NR8383细胞保护作用。从图3中可以看出PM2.5处理的细胞的存活率明显降低,然而经过FMP-1、SFMP-1、CFMP-1和AFMP-1干预的细胞的存活率升高,其中SFMP-1的效果优于其它三种多糖。
图4是不同多糖对PM2.5刺激的NR8383细胞凋亡的影响。从图4中可以看出,随着浓度的增加,四种多糖均表现出浓度依赖性地降低PM2.5引起的细胞凋亡,且SFMP-1的效果更加出色。
图5是不同多糖对PM2.5诱导的NR8383细胞内炎症因子TNF-α和IL-1β释放的影响。图5显示了PM2.5可以促进TNF-α和IL-1β的大量释放从而损伤细胞,由于多糖的干预,TNF-α和IL-1β的释放量都降低了,且呈递减趋势。相比较FMP-1、CFMP-1和AMFMP-1来说,SFMP-1可以更好地减少由于PM2.5造成的细胞因子TNF-α和IL-1β的释放,进而达到更好的保护PM2.5造成的细胞损伤。
图6是SFMP-1对PM2.5诱导的细胞凋亡的影响。PM2.5促进了细胞的凋亡,当PM2.5浓度达到200μg/mL时细胞的凋亡率为22.65%,经过SFMP-1的处理可以极大地降低PM2.5造成的凋亡,同时这种作用呈现浓度依赖性降低。当SFMP-1的浓度为200μg/mL时细胞凋亡率降到最低,为8.06%。
图7是SFMP-1对PM2.5诱导的ROS的产生量的影响。PM2.5诱导产生的ROS的量(83.1%)远远高于空白组细胞,约是空白组ROS产生量(18%)的5倍多。而经过SFMP-1的干预后,ROS的产生量明显地减少了。当SFMP-1浓度达到200μg/mL,其ROS产生量则降低为22.4%,几乎与空白组一致。
具体实施方式
下面通过具体的实施例对本发明进行详细说明,应当理解的是,这些具体实施方式仅用来帮助理解本发明,并非对本发明的实际保护范围构成任何形式的任何限定。
实施例1:
本实施例中PM2.5致肺损伤拮抗剂的制备方法如下:
1、取100mg干燥的羊肚菌多糖FMP-1置于洁净干燥的三颈烧瓶,加入甲酰胺5mL,25℃下水浴30min,随后加入200mg SO3-Py,70℃下油浴搅拌反应3h;
2、用4mol/L的NaOH溶液调步骤1获得的反应液的pH值至中性,置于3500Da透析袋中,流水透析48h,所得透析液浓缩,加入四倍体积的95vt%乙醇,4℃下静置12h,离心,收集沉淀,所得沉淀经冷冻干燥(温度-50℃,气压0.1mbar,时间72h)得到羊肚菌硫酸化多糖SFMP-1。SFMP-1的结果表征见图1(HPLC检测图)和图2(红外光谱图)。
实施例2:拮抗PM2.5损伤、保护肺泡细胞巨噬细胞实验测试
1、NR8383细胞损伤模型的建立
等到细胞长到60~80%,将细胞悬液调整以1×105cells/孔的密度种至96孔板中,每孔加入50μL的培养基,培养24h;称取15mg的PM2.5样品,悬浮于3mL的PBS中,制成5mg/mL的PM2.5悬浮液,超声使其均匀分散,用灭菌锅进行120℃,1h灭菌处理,每次使用PM2.5样品之前超声分散10min;分别将PM2.5母液稀释成50,100,200,400,600,800μg/mL的浓度加入96孔板中培养4个小时;4个小时之后每孔再加入10μL浓度为5mg/mL的MTT溶液继续培养4h;4个小时之后每孔加入100μL的酸化异丙醇(SDS、异丙醇、10mM盐酸)溶解MTT的产物,570nm波长下测量并记录其吸光值。选择细胞存活率达到50%时的PM2.5浓度建立PM2.5诱导的NR8383细胞损伤模型。
2、SFMP-1对PM2.5诱导的NR8383细胞损伤的保护作用的比较
将细胞密度调整至1×105cells/孔种入96孔板中,每孔加入50μL的细胞悬液;24个小时之后,将PM2.5母液5mg/mL稀释成200μg/mL加入到每孔中在细胞培养箱中培养4个小时;4个小时之后,SFMP-1的浓度50,100,200,400,600,800μg/mL,分别加入到每个孔中,每孔加50μL的多糖稀释液,继续相同条件培养24个小时;培养结束后,向每个孔中加入100μL的酸化异丙醇溶解甲瓒,避光震荡10min,以保证充分溶解。然后进行酶标仪570nm波长的检测。结果如图3。
3、SFMP-1对PM2.5引起的NR8383细胞的TNF-α、IL-1β生物学活性的检测
将生长状况良好,存活率>95%的L929细胞用PBS洗涤两遍,加入胰酶37℃消化细胞2分钟;加入适量含有血清的培养基中止胰酶消化,1000rpm,5min离心收集细胞,倒掉上清除去胰酶;将一定密度细胞悬液加至96孔细胞培养板中;加入不同比例的IL-1β和TNF-α标准品的稀释液,TNF-α的释液中加入1μg/mL的放线菌素D,同时收集上述步骤PM2.5和SFMP-1处理好的NR8383细胞的上清液,加入96孔板中,每孔100μL;5%CO2、37℃培养箱继续培养4h;向96孔板中加入酸化异丙醇,室温震荡10min,酶标仪570nm波长检测吸光值。结果如图4和图5。
4、SFMP-1对PM2.5诱导的NR8383细胞凋亡的保护作用
同上述步骤分别用PM2.5和SFMP-1处理细胞后,收集并洗涤细胞,按照操作步骤,进行流式细胞仪检测。结果如图6。
5、SFMP-1对PM2.5损伤的NR8383细胞ROS产生量的作用
待细胞长到一定密度收集细胞按照1×105个/孔种至六孔板中,37℃,5%CO2的环境中培养24个小时。加入浓度为200μg/mL的PM2.5培养4个小时后再加入200μ/mL的SFMP-1培养24个小时。24个小时之后,用事先预冷好的PBS洗三遍,加入10μM的DCFH-DA避光反应30min,上流式细胞仪波长488nm进行检测。结果如图7。
比较例1:
取干燥后的羊肚菌子实体,经热水浸提,醇沉除蛋白,透析(MW=3.5kDa),冷冻干燥,得羊肚菌粗多糖。取纯度为90%以上的粗多糖,通过超滤膜(MW=5kDa)超滤,Superdex-75分子筛柱层析,经冷冻干燥得羊肚菌多糖均一组分FMP-1。其活性检测步骤同实施例2,结果见图3-5。
比较例2:
取100mg羊肚菌多糖FMP-1,加入10mL异丙醇,室温下磁力搅拌器搅拌30min,加入2mL 2%NaOH溶液,剧烈搅拌1h,随后加入30mg氯乙酸,60℃反应3h。冷却至室温,用冰乙酸将pH调至中性,去离子水透析三天,然后浓缩、冷冻干燥,得羧甲基化羊肚菌多糖CMFMP-1。其活性检测步骤同实施例2,结果见图3-5。
比较例3:
取100mg羊肚菌多糖FMP-1,加入10mL去离子水,室温下磁力搅拌器搅拌30min,加入2mL 2%NaOH溶液,剧烈搅拌1h,随后逐滴加入2mL乙酸酐,室温反应1h。调pH值至中性,去离子水透析三天,然后浓缩、冷冻干燥,得乙酰化羊肚菌多糖AFMP-1。其活性检测步骤同实施例2,结果见图3-5。
Claims (6)
1.一种PM2.5致肺损伤拮抗剂,其特征在于:所述PM2.5致肺损伤拮抗剂为羊肚菌硫酸化多糖SFMP-1,分子量为9.0kDa,硫酸根取代度为1.36,其单糖组成包括甘露糖、葡萄糖和半乳糖。
2.根据权利要求1所述的PM2.5致肺损伤拮抗剂,其特征在于:
甘露糖、葡萄糖和半乳糖的摩尔比为1:7.84:1.24。
3.根据权利要求1所述的PM2.5致肺损伤拮抗剂的制备方法,其特征在于包括如下步骤:
步骤1:取100mg干燥的羊肚菌多糖FMP-1置于洁净干燥的三颈烧瓶,加入甲酰胺5mL,25℃下水浴30min,随后加入SO3-Py,70℃下搅拌反应3h;
步骤2:用NaOH溶液调步骤1获得的反应液的pH值至中性,透析,所得透析液浓缩,加入四倍体积的95vt%乙醇,4℃下静置12h,离心,收集沉淀,所得沉淀经冷冻干燥得到羊肚菌硫酸化多糖SFMP-1。
4.根据权利要求3所述的制备方法,其特征在于:
步骤1中,SO3-Py的添加量为200mg。
5.根据权利要求3所述的制备方法,其特征在于:
步骤2中,所述透析是采用3500Da透析袋,流水透析48h。
6.根据权利要求3所述的制备方法,其特征在于:
步骤2中,所述冷冻干燥的参数为:温度-50℃,气压0.1mbar,时间72h。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201810499088.4A CN108815180A (zh) | 2018-05-23 | 2018-05-23 | 一种pm2.5致肺损伤拮抗剂及其制备方法 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201810499088.4A CN108815180A (zh) | 2018-05-23 | 2018-05-23 | 一种pm2.5致肺损伤拮抗剂及其制备方法 |
Publications (1)
Publication Number | Publication Date |
---|---|
CN108815180A true CN108815180A (zh) | 2018-11-16 |
Family
ID=64148409
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN201810499088.4A Pending CN108815180A (zh) | 2018-05-23 | 2018-05-23 | 一种pm2.5致肺损伤拮抗剂及其制备方法 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN108815180A (zh) |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN102174414A (zh) * | 2010-12-28 | 2011-09-07 | 青海大学 | 一种新的肋脉羊肚菌m8-13液体发酵物在保健品及医药开发中的应用 |
CN107048349A (zh) * | 2017-03-20 | 2017-08-18 | 昆明理工大学 | 一种复方羊肚菌菌丝体多糖颗粒剂 |
-
2018
- 2018-05-23 CN CN201810499088.4A patent/CN108815180A/zh active Pending
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN102174414A (zh) * | 2010-12-28 | 2011-09-07 | 青海大学 | 一种新的肋脉羊肚菌m8-13液体发酵物在保健品及医药开发中的应用 |
CN107048349A (zh) * | 2017-03-20 | 2017-08-18 | 昆明理工大学 | 一种复方羊肚菌菌丝体多糖颗粒剂 |
Non-Patent Citations (4)
Title |
---|
WAN LI ET AL.: "Anti-inflammatory effects of Morchella esculenta polysaccharide and its derivatives in fine particulate matter-treated NR8383 cells", 《INTERNATIONAL JOURNAL OF BIOLOGICAL MACROMOLECULES》 * |
ZHENG-NAN CAI ET AL.: "Effect of polysaccharide FMP-1 from Morchella esculenta on melanogenesis in B16F10 cells and zebrafish", 《FOOD & FUNCTION》 * |
ZHENG-NAN CAI ET AL.: "Structural characterization, in vitro and in vivo antioxidant activities of a heteropolysaccharide from the fruiting bodies of Morchella esculenta", 《CARBOHYDRATE POLYMERS》 * |
陈金龙: "化学修饰羊肚菌多糖生物活性研究", 《中国优秀硕士学位论文全文数据库 工程科技辑》 * |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
Wang et al. | Sulfated modification of polysaccharides: Synthesis, characterization and bioactivities | |
CN106214694A (zh) | 瓜蒌皮多糖的应用和瓜蒌皮多糖泡腾片及其制备方法 | |
CN106749751A (zh) | 一种具有抗肝癌提高免疫力作用的香菇多糖及其制备方法 | |
CN102408494B (zh) | 一种灰树花多糖zzk组分及其制备方法 | |
Zhang et al. | Preparation and structural characterization of acid-extracted polysaccharide from Grifola frondosa and antitumor activity on S180 tumor-bearing mice | |
CN108815180A (zh) | 一种pm2.5致肺损伤拮抗剂及其制备方法 | |
EP4245304A1 (en) | Use of hyaluronic acid or salt thereof and/or trehalose in stabilizing ergothioneine, and ergothioneine composition containing hyaluronic acid or salt thereof and/or trehalose | |
CN110196333B (zh) | 一种青鳞鱼糖蛋白的提取方法和应用 | |
CN103755827A (zh) | 粒毛盘菌胞外多糖羧甲基化衍生物及其制抗肾衰药的用途 | |
CN115160450B (zh) | 鳞杯伞多糖的快速制备方法及其应用 | |
CN104725521A (zh) | 一种皱盖假芝单峰多糖f212及其制备方法和应用 | |
CN108314745B (zh) | 一种制备桦褐孔菌多糖的方法 | |
CN110051647A (zh) | 一种猴头菇多糖螯合锌微胶囊及其制备方法 | |
CN106666707B (zh) | 方格星虫抗氧化提取物的制备方法和用途 | |
CN106214799B (zh) | 一种橘皮总黄酮的提取方法 | |
CN108892732A (zh) | 具有免疫调节功能的金昌枣多糖的制备方法及其应用 | |
CN106916232B (zh) | 烟色烟管菌菌丝体多糖及其制备方法和应用 | |
CN108059652B (zh) | 一种利用蛋白酶解法制备的灰树花硒螯合肽 | |
CN106177499A (zh) | 一种铁皮石斛总黄酮提取方法 | |
CN112778430A (zh) | 一种硫酸化修饰青钱柳多糖及其制备方法和应用 | |
CN115746157B (zh) | 一种美味扇菇多糖及其制备方法和用途 | |
CN111744439B (zh) | 一种海鲜菇多糖螯合锌微胶囊及其制备方法 | |
CN110542664A (zh) | 一种检测藤茶中总黄酮类化合物含量的方法 | |
CN105002230B (zh) | 一种酶提取紫锥菊多糖的方法 | |
CN108815209A (zh) | 利用响应面法提取黑松中松多酚的方法 |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PB01 | Publication | ||
PB01 | Publication | ||
SE01 | Entry into force of request for substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
RJ01 | Rejection of invention patent application after publication | ||
RJ01 | Rejection of invention patent application after publication |
Application publication date: 20181116 |