CN115010787B - 一种苦荞活性肽及其制备方法和新用途 - Google Patents
一种苦荞活性肽及其制备方法和新用途 Download PDFInfo
- Publication number
- CN115010787B CN115010787B CN202210069362.0A CN202210069362A CN115010787B CN 115010787 B CN115010787 B CN 115010787B CN 202210069362 A CN202210069362 A CN 202210069362A CN 115010787 B CN115010787 B CN 115010787B
- Authority
- CN
- China
- Prior art keywords
- tartary buckwheat
- active peptide
- peptide
- buckwheat active
- leu
- 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.)
- Active
Links
- 108090000765 processed proteins & peptides Proteins 0.000 title claims abstract description 63
- 244000130270 Fagopyrum tataricum Species 0.000 title claims abstract description 49
- 235000014693 Fagopyrum tataricum Nutrition 0.000 title claims abstract description 49
- 238000002360 preparation method Methods 0.000 title abstract description 9
- 239000003814 drug Substances 0.000 claims abstract description 8
- 229940079593 drug Drugs 0.000 claims abstract description 6
- 206010067125 Liver injury Diseases 0.000 claims description 16
- 231100000753 hepatic injury Toxicity 0.000 claims description 14
- 230000001476 alcoholic effect Effects 0.000 claims description 12
- 125000003275 alpha amino acid group Chemical group 0.000 claims 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N EtOH Substances CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 abstract description 24
- 230000000694 effects Effects 0.000 abstract description 20
- 208000022309 Alcoholic Liver disease Diseases 0.000 abstract description 11
- 239000007788 liquid Substances 0.000 abstract description 10
- 238000000855 fermentation Methods 0.000 abstract description 8
- 230000004151 fermentation Effects 0.000 abstract description 8
- 238000000926 separation method Methods 0.000 abstract description 6
- 241000131386 Aspergillus sojae Species 0.000 abstract description 5
- 230000003110 anti-inflammatory effect Effects 0.000 abstract description 5
- 230000003078 antioxidant effect Effects 0.000 abstract description 4
- 238000012216 screening Methods 0.000 abstract description 4
- 238000004885 tandem mass spectrometry Methods 0.000 abstract description 4
- 108090000854 Oxidoreductases Proteins 0.000 abstract description 3
- 102000004316 Oxidoreductases Human genes 0.000 abstract description 3
- 229920005654 Sephadex Polymers 0.000 abstract description 3
- 239000012507 Sephadex™ Substances 0.000 abstract description 3
- 239000003963 antioxidant agent Substances 0.000 abstract description 3
- 230000015572 biosynthetic process Effects 0.000 abstract description 3
- 238000000338 in vitro Methods 0.000 abstract description 3
- 238000003786 synthesis reaction Methods 0.000 abstract description 3
- 239000012045 crude solution Substances 0.000 abstract description 2
- 239000002994 raw material Substances 0.000 abstract description 2
- 238000000108 ultra-filtration Methods 0.000 abstract description 2
- 238000000746 purification Methods 0.000 abstract 1
- 239000007787 solid Substances 0.000 abstract 1
- 238000009210 therapy by ultrasound Methods 0.000 abstract 1
- 241000699670 Mus sp. Species 0.000 description 18
- 239000000243 solution Substances 0.000 description 17
- 230000001154 acute effect Effects 0.000 description 9
- WSMYVTOQOOLQHP-UHFFFAOYSA-N Malondialdehyde Chemical compound O=CCC=O WSMYVTOQOOLQHP-UHFFFAOYSA-N 0.000 description 8
- 210000004185 liver Anatomy 0.000 description 8
- 229940118019 malondialdehyde Drugs 0.000 description 8
- 102000004196 processed proteins & peptides Human genes 0.000 description 8
- 238000001514 detection method Methods 0.000 description 7
- 210000005228 liver tissue Anatomy 0.000 description 7
- 210000002966 serum Anatomy 0.000 description 7
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 7
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 6
- 210000005229 liver cell Anatomy 0.000 description 6
- 239000006228 supernatant Substances 0.000 description 5
- 241001465754 Metazoa Species 0.000 description 4
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 description 4
- 150000001413 amino acids Chemical group 0.000 description 4
- 238000010828 elution Methods 0.000 description 4
- 238000002474 experimental method Methods 0.000 description 4
- HJUFTIJOISQSKQ-UHFFFAOYSA-N fenoxycarb Chemical compound C1=CC(OCCNC(=O)OCC)=CC=C1OC1=CC=CC=C1 HJUFTIJOISQSKQ-UHFFFAOYSA-N 0.000 description 4
- 230000003908 liver function Effects 0.000 description 4
- 230000002829 reductive effect Effects 0.000 description 4
- 239000008223 sterile water Substances 0.000 description 4
- 210000001519 tissue Anatomy 0.000 description 4
- 241000699666 Mus <mouse, genus> Species 0.000 description 3
- 239000008280 blood Substances 0.000 description 3
- 210000004369 blood Anatomy 0.000 description 3
- 230000003203 everyday effect Effects 0.000 description 3
- 230000017074 necrotic cell death Effects 0.000 description 3
- 235000018102 proteins Nutrition 0.000 description 3
- 108090000623 proteins and genes Proteins 0.000 description 3
- 102000004169 proteins and genes Human genes 0.000 description 3
- 108010021809 Alcohol dehydrogenase Proteins 0.000 description 2
- 101710088194 Dehydrogenase Proteins 0.000 description 2
- 241000219051 Fagopyrum Species 0.000 description 2
- 235000009419 Fagopyrum esculentum Nutrition 0.000 description 2
- ONIBWKKTOPOVIA-BYPYZUCNSA-N L-Proline Chemical compound OC(=O)[C@@H]1CCCN1 ONIBWKKTOPOVIA-BYPYZUCNSA-N 0.000 description 2
- QNAYBMKLOCPYGJ-REOHCLBHSA-N L-alanine Chemical compound C[C@H](N)C(O)=O QNAYBMKLOCPYGJ-REOHCLBHSA-N 0.000 description 2
- ROHFNLRQFUQHCH-YFKPBYRVSA-N L-leucine Chemical compound CC(C)C[C@H](N)C(O)=O ROHFNLRQFUQHCH-YFKPBYRVSA-N 0.000 description 2
- ROHFNLRQFUQHCH-UHFFFAOYSA-N Leucine Natural products CC(C)CC(N)C(O)=O ROHFNLRQFUQHCH-UHFFFAOYSA-N 0.000 description 2
- ONIBWKKTOPOVIA-UHFFFAOYSA-N Proline Natural products OC(=O)C1CCCN1 ONIBWKKTOPOVIA-UHFFFAOYSA-N 0.000 description 2
- PMZXXNPJQYDFJX-UHFFFAOYSA-N acetonitrile;2,2,2-trifluoroacetic acid Chemical compound CC#N.OC(=O)C(F)(F)F PMZXXNPJQYDFJX-UHFFFAOYSA-N 0.000 description 2
- 235000004279 alanine Nutrition 0.000 description 2
- 210000004027 cell Anatomy 0.000 description 2
- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 description 2
- 238000012258 culturing Methods 0.000 description 2
- 230000006378 damage Effects 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 239000000706 filtrate Substances 0.000 description 2
- HQVFCQRVQFYGRJ-UHFFFAOYSA-N formic acid;hydrate Chemical compound O.OC=O HQVFCQRVQFYGRJ-UHFFFAOYSA-N 0.000 description 2
- 238000004108 freeze drying Methods 0.000 description 2
- 230000036541 health Effects 0.000 description 2
- 238000004128 high performance liquid chromatography Methods 0.000 description 2
- 230000006872 improvement Effects 0.000 description 2
- 230000002401 inhibitory effect Effects 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 230000004060 metabolic process Effects 0.000 description 2
- 210000004940 nucleus Anatomy 0.000 description 2
- 230000036961 partial effect Effects 0.000 description 2
- 239000002504 physiological saline solution Substances 0.000 description 2
- 239000013641 positive control Substances 0.000 description 2
- 239000000843 powder Substances 0.000 description 2
- 238000012163 sequencing technique Methods 0.000 description 2
- 238000010563 solid-state fermentation Methods 0.000 description 2
- 239000000725 suspension Substances 0.000 description 2
- 238000002137 ultrasound extraction Methods 0.000 description 2
- XXMFJKNOJSDQBM-UHFFFAOYSA-N 2,2,2-trifluoroacetic acid;hydrate Chemical compound [OH3+].[O-]C(=O)C(F)(F)F XXMFJKNOJSDQBM-UHFFFAOYSA-N 0.000 description 1
- 208000021959 Abnormal metabolism Diseases 0.000 description 1
- 102000007698 Alcohol dehydrogenase Human genes 0.000 description 1
- 206010003694 Atrophy Diseases 0.000 description 1
- 206010006187 Breast cancer Diseases 0.000 description 1
- 208000026310 Breast neoplasm Diseases 0.000 description 1
- 206010019670 Hepatic function abnormal Diseases 0.000 description 1
- 206010019799 Hepatitis viral Diseases 0.000 description 1
- 208000031226 Hyperlipidaemia Diseases 0.000 description 1
- COLNVLDHVKWLRT-QMMMGPOBSA-N L-phenylalanine Chemical compound OC(=O)[C@@H](N)CC1=CC=CC=C1 COLNVLDHVKWLRT-QMMMGPOBSA-N 0.000 description 1
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 1
- 239000004472 Lysine Substances 0.000 description 1
- 229930040373 Paraformaldehyde Natural products 0.000 description 1
- 241000219050 Polygonaceae Species 0.000 description 1
- 231100000439 acute liver injury Toxicity 0.000 description 1
- 230000032683 aging Effects 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 238000010171 animal model Methods 0.000 description 1
- 230000003064 anti-oxidating effect Effects 0.000 description 1
- 230000006907 apoptotic process Effects 0.000 description 1
- 230000037444 atrophy Effects 0.000 description 1
- 230000037208 balanced nutrition Effects 0.000 description 1
- 235000019046 balanced nutrition Nutrition 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 235000010290 biphenyl Nutrition 0.000 description 1
- 239000004305 biphenyl Substances 0.000 description 1
- 125000006267 biphenyl group Chemical group 0.000 description 1
- 210000001124 body fluid Anatomy 0.000 description 1
- 239000010839 body fluid Substances 0.000 description 1
- 230000037396 body weight Effects 0.000 description 1
- 210000005252 bulbus oculi Anatomy 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 235000012000 cholesterol Nutrition 0.000 description 1
- 239000012043 crude product Substances 0.000 description 1
- 230000002354 daily effect Effects 0.000 description 1
- 206010012601 diabetes mellitus Diseases 0.000 description 1
- 230000008034 disappearance Effects 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000035475 disorder Diseases 0.000 description 1
- 230000035622 drinking Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 235000020776 essential amino acid Nutrition 0.000 description 1
- 239000003797 essential amino acid Substances 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 230000006870 function Effects 0.000 description 1
- 208000001130 gallstones Diseases 0.000 description 1
- 230000002496 gastric effect Effects 0.000 description 1
- 238000005227 gel permeation chromatography Methods 0.000 description 1
- 231100000234 hepatic damage Toxicity 0.000 description 1
- 239000012510 hollow fiber Substances 0.000 description 1
- 201000001421 hyperglycemia Diseases 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 210000004969 inflammatory cell Anatomy 0.000 description 1
- 230000028709 inflammatory response Effects 0.000 description 1
- 230000008818 liver damage Effects 0.000 description 1
- 208000019423 liver disease Diseases 0.000 description 1
- 238000011068 loading method Methods 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000006371 metabolic abnormality Effects 0.000 description 1
- 238000000034 method Methods 0.000 description 1
- 238000003032 molecular docking Methods 0.000 description 1
- 238000010172 mouse model Methods 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 230000035764 nutrition Effects 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 239000012188 paraffin wax Substances 0.000 description 1
- 229920002866 paraformaldehyde Polymers 0.000 description 1
- 231100000915 pathological change Toxicity 0.000 description 1
- 230000036285 pathological change Effects 0.000 description 1
- 230000007170 pathology Effects 0.000 description 1
- COLNVLDHVKWLRT-UHFFFAOYSA-N phenylalanine Natural products OC(=O)C(N)CC1=CC=CC=C1 COLNVLDHVKWLRT-UHFFFAOYSA-N 0.000 description 1
- ZUOUZKKEUPVFJK-UHFFFAOYSA-N phenylbenzene Natural products C1=CC=CC=C1C1=CC=CC=C1 ZUOUZKKEUPVFJK-UHFFFAOYSA-N 0.000 description 1
- 229920002492 poly(sulfone) Polymers 0.000 description 1
- 230000002035 prolonged effect Effects 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 230000001681 protective effect Effects 0.000 description 1
- 238000010791 quenching Methods 0.000 description 1
- 230000011506 response to oxidative stress Effects 0.000 description 1
- 238000004007 reversed phase HPLC Methods 0.000 description 1
- 210000000952 spleen Anatomy 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 230000000638 stimulation Effects 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 210000002784 stomach Anatomy 0.000 description 1
- 238000005728 strengthening Methods 0.000 description 1
- 230000035922 thirst Effects 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 201000001862 viral hepatitis Diseases 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
-
- 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
- A23L33/17—Amino acids, peptides or proteins
- A23L33/18—Peptides; Protein hydrolysates
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
- C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
-
- 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
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- General Health & Medical Sciences (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Wood Science & Technology (AREA)
- Biochemistry (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Zoology (AREA)
- General Engineering & Computer Science (AREA)
- Animal Behavior & Ethology (AREA)
- Biotechnology (AREA)
- Biophysics (AREA)
- Gastroenterology & Hepatology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Pharmacology & Pharmacy (AREA)
- Microbiology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Mycology (AREA)
- Nutrition Science (AREA)
- Food Science & Technology (AREA)
- Polymers & Plastics (AREA)
- Medicines Containing Plant Substances (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
本发明公开一种苦荞活性肽及其制备方法和新用途，即苦荞活性肽在防治酒精性肝病药物中的应用。该苦荞活性肽的肽序为Lys‑Leu‑Arg‑Pro‑Leu‑Gln‑Val‑Leu，制备方法包括：以苦荞为原料经酱油曲霉固态发酵、超声处理后得苦荞活性肽粗溶液，依次进行超滤处理、葡聚糖凝胶色谱柱分离和高效液相色谱柱纯化，经Q‑ExactiveLC‑MS/MS进行序列鉴定后初步筛选，筛选后的肽与乙醇氧化酶分子对接后再次筛选进行合成，经体外抗氧化抗炎活性测定后选取活性效果最好的一种肽。经过研究发现所述的苦荞活性肽用于对酒精性肝病的治疗。
Description
技术领域
本发明涉及一种苦荞活性肽及其制备方法和新用途，尤其涉及一种苦荞活性肽及其制备和在治疗酒精性肝病药物中的应用，属于食品与医药技术领域。
背景技术
酒精性肝病(alcoholic liver disease,ALD)是长期过量饮酒引起的慢性肝病。在全世界范围内,酒精性肝病即病毒性肝炎之后,成为肝脏损害的第二大主要病因。近年来我国酒精性疾病发病率呈增长趋势,严重影响患者日常生活、工作,对其生命健康造成威胁。目前对于酒精性肝病无特效药物，主要治疗仍以戒酒为主。
苦荞(Fagopyrum tataricum)起源于中国和亚洲北部地区，为蓼科(Polygonaceae)荞麦属(Fagopyrum)一年生双子叶作物，具有生育期短、耐冷凉、耐贫瘠、适应性较强等特点，常作为山区、丘陵区轮作换茬的重要作物之一；苦荞含有丰富的营养和保健成分，尤其是在预防糖尿病、高血糖、高血脂等方面有重要作用；其中，苦荞的蛋白质含量为13.4%，富含赖氨酸、苯丙氨酸等多种必需氨基酸，是一种营养均衡的蛋白质，可以很好地为人体所利用。有研究发现苦荞蛋白具有降低胆固醇、降血脂、抗衰老、抑制乳腺癌的发生、抑制胆结石等作用；但是将苦荞活性肽用于治疗酒精性肝病尚未被报道。
发明内容
本发明针对现有技术中存在的问题，本发明的目的一在于提供具有新型氨基酸序列的苦荞活性肽，所述苦荞活性肽的氨基酸序列为：Lys-Leu-Arg-Pro-Leu-Gln-Val-Leu，经动物试验发现，该苦荞活性肽能够显著减轻酒精导致的肝损伤，可用于制备治疗酒精性肝病的药物。
本发明的目的二在于提供所述苦荞活性肽在制备治疗肝损伤药物中的应用。
本发明的目的四在于提供所述苦荞活性肽的制备方法，具体包括以下步骤：
（1）以苦荞为原料，经酱油曲霉固态发酵后超声提取制备得苦荞活性肽粗液。
（2）采用截留分子量为5000Da的超滤膜（例如中空纤维聚砜超滤膜）对苦荞活性肽粗液进行超滤，收集小于5000Da的超滤液冷冻干燥，备用。
（3）以水为流动相（流速优选0.3mL/min），采用葡聚糖凝胶（例如G25凝胶）对步骤（2）得到的苦荞活性肽进行粗分。
（4）利用高效液相色谱技术对步骤（3）得到的粗分产物进一步分离，多次收集合并，冷冻干燥备用。
（5）将步骤（4）收集的苦荞活性肽复溶，经Q-ExactiveLC-MS/MS进行序列鉴定后进行初步筛选，根据测序结果选择含亮氨酸、脯氨酸和丙氨酸三种氨基酸中两种及以上的肽序列。
（6）将步骤（5）所得的肽与乙醇氧化酶分子对接后再次筛选进行合成。
（7）将合成后的肽经体外抗氧化抗炎活性测定，选取活性最好的肽即为所述苦荞活性肽。
优选的，本发明步骤（1）中酱油曲霉固态发酵具体过程为：将10-20g苦荞粉经高压灭菌后加入1-3mL的酱油曲霉孢子悬液（2×107个/mL）和2-10mL无菌水，搅拌均匀后放入温度为20-40℃、湿度为60%-90%的环境中培养20-40h；发酵结束后向发酵罐中加入20-60mL无菌水，超声提取10-40min，离心取上清液，即为苦荞粗溶液。
优选的，本发明步骤（4）中所述的高效液相色谱的分离条件为：
色谱柱：Angilent Eclipse XDB-C18柱。
流动相:A液：0.1％三氟乙酸-水溶液；B液：0.1％三氟乙酸-乙腈溶液。
采用线性梯度洗脱：0-10min，10％～30％的B液；10～30min，30％～50％的B液；30-50min，50-80%的B液。
流速为0.6mL/min，柱温为30℃，检测波长为220nm。
优选的，本发明步骤（4）中所述Q-ExactiveLC-MS/MS分离条件为：
色谱柱：Angilent Eclipse XDB-C18柱。
流动相：A液：0.1％甲酸水；B液：纯乙腈。
采用线性梯度洗脱：0-10min，5％～25％B液；10～40min，25％～75％B液。
流速为0.3mL/min，柱温为30℃，检测波长为220nm。
除特殊说明外，本发明所有百分比均为质量百分比。
本发明所提供的解酒护肝的制剂，能通过加快酒精的代谢达到醒酒和解酒的效果，且其还能改善酒精导致的新陈代谢的异常、调节人体机能，具有清热解毒、生津止渴和健脾胃护肝的效果从而降低了酒精性肝损伤的发生。
本发明的有益效果
本发明公开了一种具有新型氨基酸序列结构的活性肽；经活性试验发现，该活性肽具有良好的抗氧化和抗炎活性，能够显著改善酒精性肝病模型小鼠的氧化应激和炎症反应，可用于制备治疗酒精性肝病的药物。
附图说明
图1为苦荞活性肽对急性酒精性肝损伤小鼠组织病理学的影响；
图2为苦荞活性肽对急性酒精性肝损伤小鼠肝功能的影响；
图3为苦荞活性肽对急性酒精性肝损伤小鼠体内丙二醛含量的影响。
具体实施方式
下面结合具体实施例，对本发明的实施例技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例；基于本发明的实施例，本领域普通技术人员在没有作出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
实施例1
本实施例所述苦荞活性肽的制备方法，具体包括以下步骤：
（1）苦荞粉10g装入发酵罐中，121℃灭菌20min；加入酱油曲霉孢子悬液（2×107个/mL）2.47mL、无菌水6.04mL于温度为28℃、湿度为80%的环境中培养39.84h；发酵结束后在超净台向发酵罐中加入50mL无菌水，并在功率200W，温度为18℃超声机中提取30min，而后在4℃、10000r/min条件下离心15min，收集上清液。
（2）将滤液用截留分子量为5000Da的空纤维聚砜超滤膜进行超滤，取过滤液，并对其进行冷冻干燥，得到分子量<5000Da的苦荞活性肽；将冷冻干燥的苦荞活性肽复溶成30mg/mL的肽溶液，过0.22μm水膜，取200μL溶液上葡聚糖凝胶G25色谱柱，控制柱流速0.3mL/min。
（3）利用RP-HPLC技术分别分离凝胶层析分离得到组分，分离条件为：色谱柱：Angilent Eclipse XDB-C18柱(4×250mm，5μm)流动相：A液：0.1％三氟乙酸(TFA)水溶液，B液：0.1％TFA-乙腈溶液，线性梯度洗脱，0-10min，10％-30％B液；10-30min，30％-50％B液，流速为0.6mL/min，柱温：30℃，检测波长：220nm。
（4）经Q-ExactiveLC-MS/MS分析，分离条件为：色谱柱：Angilent Eclipse XDB-C18柱；流动相：A液：0.1％甲酸水；B液：纯乙腈；采用线性梯度洗脱：0-10min，5％～25％B液；10～40min，25％～75％B液，流速为0.3mL/min，柱温为30℃，检测波长为220nm。分析所得的肽根据测序结果选择亮氨酸、脯氨酸和丙氨酸含量高的肽序列。
（5）将筛选所得24种肽在软件SYBYL软件进行构建优化，然后与乙醇氧化酶进行分子对接，筛选出T-评分≥10的肽，得到8种肽，合成后进行体外抗氧化抗炎活性测定，筛选活性效果最好的一种活性肽，该活性肽肽序为Lys-Leu-Arg-Pro-Leu-Gln-Val-Leu，如表1和表2所示。
表1.八种肽的抗氧化活性
表2.八种肽的抗炎活性
实施例2
本实施例通过动物实验进一步证明该苦荞活性肽（氨基酸序列为：Lys-Leu-Arg-Pro-Leu-Gln-Val-Leu）对急性酒精性肝损伤小鼠的干预和改善作用，具体实验如下：
1小鼠急性酒精性肝损伤的诱导与干预
将32只C57BL/6小鼠（20-22g）(由昆明医科大学动物学部提供)，适应性饲喂养1周后，随机分为4组，每组8只，分别分为正常组、模型组、阳性对照组、苦荞活性肽治疗组。阳性对照组每天按照200mg/kg体重灌胃联苯双脂，苦荞活性肽治疗组每天按照4mg/kg体重灌胃苦荞活性肽，对照组和模型组每天灌胃相同体积生理盐水，实验持续21天。末次给药后2h，除对照组外，其余小鼠按12mL/kg体重灌胃56%乙醇诱导急性酒精性肝损伤小鼠模型。
2动物模型指标
2.1小鼠肝脏组织病理学检测
完成酒精灌胃后，小鼠禁食不禁水，12 h后眼球取血，脱颈处死小鼠，在超净台上迅速摘取肝脏并记录其重量，并取部分组织浸泡于10%多聚甲醛固定液中，常规石蜡包埋，H＆E染色，于光学显微镜下观察肝脏组织的病理学改变。
2.2小鼠肝功能检测
收集血样，在4000 r，4 ℃下离心10 min，取上清，按试剂盒说明书检测血清AST、ALT水平。
称取肝脏组织，用生理盐水匀化10%的肝脏组织；在4 ℃下以2500 r /min的速度离心10 min后，收集肝脏均质的上清液；按试剂盒说明书检测（乙醇脱氢酶）ADH水平。
2.3肝脏组织中丙二醛（MDA）水平检测
基于上述收集的小鼠血清和肝脏组织均质的上清液，分别按照试剂盒说明书测定小鼠血清和肝脏组织中MDA含量。
本实施例中，结果用Mean±SD表示，数据选用SPSS 13.0统计软件进行分析，以P<0.05表示统计学差异显著。
3.附图分析及结论如下
3.1苦荞活性肽对进行酒精性肝损伤小鼠肝脏组织病理的影响
对照组细胞排列整齐，肝小叶结构完整，肝细胞核及细胞边界清晰，未见坏死。模型组肝细胞排列紊乱，肝细胞间隙有炎性细胞浸润，肝细胞肿胀，肝细胞核部分萎缩，甚至消失或部分坏死，有明显脂肪滴，说明造模成功，急性酒精刺激对小鼠肝脏造成了较为严重的损伤；给予苦荞活性肽治疗后，能够显著改善肝细胞凋亡坏死现象，说明苦荞活性肽对酒精诱导的小鼠急性肝损伤有保护作用。
3.2苦荞活性肽对急性酒精性肝损伤小鼠肝功能的影响
如图2，与对照组相比，模型组血清ALT(P<0.05)与AST(P<0.05)含量明显升高，说明过量食用酒精导致小鼠肝功能损伤。与模型组比较，苦荞活性肽治疗组血清中ALT与AST含量明显降低。可以看出模型组乙醇脱氢酶含量显著降低（p＜0.05），这是由于短期大量饮酒导致肝脏酒精代谢紊乱，造成肝损伤使得乙醇脱氢酶表达下降。相比于模型组，给药组有显著改善，说明苦荞活性肽对酒精诱导的肝功能损坏有一定改善作用。
3.3苦荞活性肽对急性酒精性肝损伤小鼠体内MDA含量的影响
如图3所示，与对照组相比，模型组小鼠的血清MDA和组织中MDA水平明显升高，与模型组相比，苦荞活性肽治疗组血清和组织中MDA水平显著降低(P<0.05)；这表明经酒精诱导后，肝脏组织氧化损伤严重而该苦荞活性肽具有改善作用。
Claims (2)
1.一种苦荞活性肽，其特征在于：所述苦荞活性肽的氨基酸序列为：Lys-Leu-Arg-Pro-Leu-Gln-Val-Leu。
2.权利要求1所述苦荞活性肽在制备治疗酒精性肝损伤药物中的应用。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210069362.0A CN115010787B (zh) | 2022-01-21 | 2022-01-21 | 一种苦荞活性肽及其制备方法和新用途 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210069362.0A CN115010787B (zh) | 2022-01-21 | 2022-01-21 | 一种苦荞活性肽及其制备方法和新用途 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN115010787A CN115010787A (zh) | 2022-09-06 |
| CN115010787B true CN115010787B (zh) | 2024-05-14 |
Family
ID=83067901
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210069362.0A Active CN115010787B (zh) | 2022-01-21 | 2022-01-21 | 一种苦荞活性肽及其制备方法和新用途 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN115010787B (zh) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117247431B (zh) * | 2023-11-17 | 2024-01-30 | 中国农业大学 | 一种具有dpp-iv抑制活性的苦荞肽及其应用 |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104312895A (zh) * | 2014-10-24 | 2015-01-28 | 三原县甘露池醋厂 | 一种苦荞醋及其制备方法 |
| CN107058433A (zh) * | 2017-05-16 | 2017-08-18 | 昆明理工大学 | 一种具有抗菌和抗氧化双活性的苦荞活性肽的制备方法 |
| CN109336953A (zh) * | 2018-11-26 | 2019-02-15 | 贵州医科大学 | 一种苦荞抗氧化肽及其制备方法和应用 |
| CN110664834A (zh) * | 2019-11-14 | 2020-01-10 | 济宁医学院 | 苦荞麦多糖制备治疗非酒精性脂肪肝药物中的应用 |
-
2022
- 2022-01-21 CN CN202210069362.0A patent/CN115010787B/zh active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104312895A (zh) * | 2014-10-24 | 2015-01-28 | 三原县甘露池醋厂 | 一种苦荞醋及其制备方法 |
| CN107058433A (zh) * | 2017-05-16 | 2017-08-18 | 昆明理工大学 | 一种具有抗菌和抗氧化双活性的苦荞活性肽的制备方法 |
| CN109336953A (zh) * | 2018-11-26 | 2019-02-15 | 贵州医科大学 | 一种苦荞抗氧化肽及其制备方法和应用 |
| CN110664834A (zh) * | 2019-11-14 | 2020-01-10 | 济宁医学院 | 苦荞麦多糖制备治疗非酒精性脂肪肝药物中的应用 |
Non-Patent Citations (1)
| Title |
|---|
| 苦荞麸皮总黄酮体外抗氧化活性及体内解酒护肝作用;童钰琴等;《食品工业科技》;第41卷(第17期);第314-319+326页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN115010787A (zh) | 2022-09-06 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| KR100189144B1 (ko) | 신규 생리 활성 물질 | |
| TWI314864B (en) | Pharmaceutical composition for treating inflammation, pain, arthritis and spinitis, and proliferating osteoblastic cell and method thereof | |
| CN115010787B (zh) | 一种苦荞活性肽及其制备方法和新用途 | |
| EP2120980B1 (en) | An anti-diabetic extract of rooibos | |
| CN112979748B (zh) | 一种具有预防慢性酒精性肝损伤作用的活性肽 | |
| US8476229B2 (en) | Blood sugar-modulating polypeptides | |
| CN104152518B (zh) | 一种肝病辅食型鳕鱼皮胶原蛋白肽的制备方法 | |
| CN110693025B (zh) | 一种多功能成分协同降血糖的组合物及其应用 | |
| CN114989248B (zh) | 一种具有抗炎抗氧化活性的忧遁草多肽及其制备方法和应用 | |
| CN114213506B (zh) | 一种人参来源的抗血管生成及抗肿瘤活性肽及其制备方法和应用 | |
| CN110959861A (zh) | 一种含有sod组份的美白口服液及其制备方法 | |
| KR100749944B1 (ko) | 여주에서 제조된 혈당강하용 사포닌 분획물 및 이를 포함하는 약제학적 조성물 | |
| CN116751247A (zh) | 具保肝护肝功效的金枪鱼心脏动脉球抗氧化肽及制备方法和应用 | |
| US20090169658A1 (en) | Toona sinensis extract for suppressing proliferation and inducing apoptosis of osteosarcoma cells | |
| KR101887764B1 (ko) | 항비만성 팥 껍질 추출물의 추출방법 및 이를 유효성분으로 함유하는 항비만 조성물 | |
| CN113694104B (zh) | 一种对化学性肝损伤有保护作用、促进肝脏再生的中药组合物、制备方法及其用途 | |
| CN113444759A (zh) | 一种乙醇脱氢酶激活肽及其制备方法 | |
| Wu et al. | In vivo antioxidant activity of black soybean peptide in aging mice caused by D-galactose | |
| CN107604032B (zh) | 一种具有抗疲劳作用的驼血多肽及其制备方法 | |
| CN113134034A (zh) | 一种中药组合物、应用及中药制剂 | |
| CN115969893B (zh) | 一种结球甘蓝总硫苷的提取方法与应用 | |
| CN116693711B (zh) | 一种何首乌多糖及其提取方法和应用 | |
| CN101081244A (zh) | 一种植物来源降血糖活性多肽口服生物活性的保存方法 | |
| CN115501294B (zh) | 一种布拉氏酵母菌发酵怀山药醇提物的制备方法及应用 | |
| CN116836223A (zh) | 一种青刺果活性肽组合物及新用途 |
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 | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |