CN113875990B - Ferrous melanin with antioxidant activity and preparation method thereof - Google Patents
Ferrous melanin with antioxidant activity and preparation method thereof Download PDFInfo
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- CN113875990B CN113875990B CN202111179686.1A CN202111179686A CN113875990B CN 113875990 B CN113875990 B CN 113875990B CN 202111179686 A CN202111179686 A CN 202111179686A CN 113875990 B CN113875990 B CN 113875990B
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- bsm
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- XUMBMVFBXHLACL-UHFFFAOYSA-N Melanin Chemical compound O=C1C(=O)C(C2=CNC3=C(C(C(=O)C4=C32)=O)C)=C2C4=CNC2=C1C XUMBMVFBXHLACL-UHFFFAOYSA-N 0.000 title claims abstract description 98
- 238000002360 preparation method Methods 0.000 title claims abstract description 18
- 230000003078 antioxidant effect Effects 0.000 title claims abstract description 14
- CWYNVVGOOAEACU-UHFFFAOYSA-N Fe2+ Chemical compound [Fe+2] CWYNVVGOOAEACU-UHFFFAOYSA-N 0.000 title claims abstract description 8
- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 claims abstract description 254
- 229910052742 iron Inorganic materials 0.000 claims abstract description 127
- 230000009920 chelation Effects 0.000 claims abstract description 62
- 238000006243 chemical reaction Methods 0.000 claims abstract description 48
- 238000000034 method Methods 0.000 claims abstract description 15
- 230000004044 response Effects 0.000 claims abstract description 15
- 238000002156 mixing Methods 0.000 claims abstract description 10
- 238000003756 stirring Methods 0.000 claims abstract description 10
- 235000007215 black sesame Nutrition 0.000 claims abstract description 9
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 7
- RBTARNINKXHZNM-UHFFFAOYSA-K iron trichloride Chemical compound Cl[Fe](Cl)Cl RBTARNINKXHZNM-UHFFFAOYSA-K 0.000 claims abstract description 5
- 239000002244 precipitate Substances 0.000 claims abstract description 5
- 238000009777 vacuum freeze-drying Methods 0.000 claims abstract description 4
- 238000005406 washing Methods 0.000 claims abstract description 4
- 230000035484 reaction time Effects 0.000 claims description 19
- 238000002474 experimental method Methods 0.000 claims description 16
- 238000011835 investigation Methods 0.000 claims description 10
- 238000013461 design Methods 0.000 claims description 7
- 238000010521 absorption reaction Methods 0.000 abstract description 17
- 206010067484 Adverse reaction Diseases 0.000 abstract description 6
- 208000015710 Iron-Deficiency Anemia Diseases 0.000 abstract description 6
- 230000006838 adverse reaction Effects 0.000 abstract description 6
- 230000036541 health Effects 0.000 abstract description 5
- 230000001105 regulatory effect Effects 0.000 abstract description 5
- 230000002349 favourable effect Effects 0.000 abstract description 3
- 239000003963 antioxidant agent Substances 0.000 abstract description 2
- NQXWGWZJXJUMQB-UHFFFAOYSA-K iron trichloride hexahydrate Chemical compound O.O.O.O.O.O.[Cl-].Cl[Fe+]Cl NQXWGWZJXJUMQB-UHFFFAOYSA-K 0.000 abstract description 2
- 230000009977 dual effect Effects 0.000 abstract 1
- 239000000243 solution Substances 0.000 description 51
- -1 iron ions Chemical class 0.000 description 24
- 230000000694 effects Effects 0.000 description 21
- 238000007254 oxidation reaction Methods 0.000 description 12
- 239000013589 supplement Substances 0.000 description 12
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- 239000000523 sample Substances 0.000 description 10
- 238000002835 absorbance Methods 0.000 description 9
- 230000003064 anti-oxidating effect Effects 0.000 description 7
- 239000013522 chelant Substances 0.000 description 7
- 230000001502 supplementing effect Effects 0.000 description 7
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 6
- OHDRQQURAXLVGJ-HLVWOLMTSA-N azane;(2e)-3-ethyl-2-[(e)-(3-ethyl-6-sulfo-1,3-benzothiazol-2-ylidene)hydrazinylidene]-1,3-benzothiazole-6-sulfonic acid Chemical compound [NH4+].[NH4+].S/1C2=CC(S([O-])(=O)=O)=CC=C2N(CC)C\1=N/N=C1/SC2=CC(S([O-])(=O)=O)=CC=C2N1CC OHDRQQURAXLVGJ-HLVWOLMTSA-N 0.000 description 6
- 239000002245 particle Substances 0.000 description 6
- 206010022971 Iron Deficiencies Diseases 0.000 description 5
- 125000003118 aryl group Chemical group 0.000 description 5
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 5
- 230000002496 gastric effect Effects 0.000 description 5
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 5
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 4
- 208000007502 anemia Diseases 0.000 description 4
- 238000004090 dissolution Methods 0.000 description 4
- 230000006870 function Effects 0.000 description 4
- 230000003993 interaction Effects 0.000 description 4
- 229910021645 metal ion Inorganic materials 0.000 description 4
- IOLCXVTUBQKXJR-UHFFFAOYSA-M potassium bromide Chemical compound [K+].[Br-] IOLCXVTUBQKXJR-UHFFFAOYSA-M 0.000 description 4
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 4
- 239000012085 test solution Substances 0.000 description 4
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- HHEAADYXPMHMCT-UHFFFAOYSA-N dpph Chemical compound [O-][N+](=O)C1=CC([N+](=O)[O-])=CC([N+]([O-])=O)=C1[N]N(C=1C=CC=CC=1)C1=CC=CC=C1 HHEAADYXPMHMCT-UHFFFAOYSA-N 0.000 description 3
- 235000011389 fruit/vegetable juice Nutrition 0.000 description 3
- 230000036039 immunity Effects 0.000 description 3
- 238000005259 measurement Methods 0.000 description 3
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- 238000005033 Fourier transform infrared spectroscopy Methods 0.000 description 2
- 206010070840 Gastrointestinal tract irritation Diseases 0.000 description 2
- WTDHULULXKLSOZ-UHFFFAOYSA-N Hydroxylamine hydrochloride Chemical compound Cl.ON WTDHULULXKLSOZ-UHFFFAOYSA-N 0.000 description 2
- 206010028980 Neoplasm Diseases 0.000 description 2
- 230000002292 Radical scavenging effect Effects 0.000 description 2
- DGEZNRSVGBDHLK-UHFFFAOYSA-N [1,10]phenanthroline Chemical compound C1=CN=C2C3=NC=CC=C3C=CC2=C1 DGEZNRSVGBDHLK-UHFFFAOYSA-N 0.000 description 2
- 150000001413 amino acids Chemical class 0.000 description 2
- 125000003277 amino group Chemical group 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 239000007853 buffer solution Substances 0.000 description 2
- 125000002915 carbonyl group Chemical group [*:2]C([*:1])=O 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 238000004140 cleaning Methods 0.000 description 2
- 206010012601 diabetes mellitus Diseases 0.000 description 2
- 238000007865 diluting Methods 0.000 description 2
- MGJZITXUQXWAKY-UHFFFAOYSA-N diphenyl-(2,4,6-trinitrophenyl)iminoazanium Chemical compound [O-][N+](=O)C1=CC([N+](=O)[O-])=CC([N+]([O-])=O)=C1N=[N+](C=1C=CC=CC=1)C1=CC=CC=C1 MGJZITXUQXWAKY-UHFFFAOYSA-N 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 238000002296 dynamic light scattering Methods 0.000 description 2
- 238000013401 experimental design Methods 0.000 description 2
- 235000003891 ferrous sulphate Nutrition 0.000 description 2
- 239000011790 ferrous sulphate Substances 0.000 description 2
- 239000000706 filtrate Substances 0.000 description 2
- 238000001914 filtration Methods 0.000 description 2
- 239000012530 fluid Substances 0.000 description 2
- 230000007760 free radical scavenging Effects 0.000 description 2
- 210000004051 gastric juice Anatomy 0.000 description 2
- 210000001035 gastrointestinal tract Anatomy 0.000 description 2
- 239000001257 hydrogen Substances 0.000 description 2
- 229910052739 hydrogen Inorganic materials 0.000 description 2
- 238000000338 in vitro Methods 0.000 description 2
- 238000002329 infrared spectrum Methods 0.000 description 2
- 230000005764 inhibitory process Effects 0.000 description 2
- 230000000968 intestinal effect Effects 0.000 description 2
- 239000002608 ionic liquid Substances 0.000 description 2
- 150000002500 ions Chemical class 0.000 description 2
- BAUYGSIQEAFULO-UHFFFAOYSA-L iron(2+) sulfate (anhydrous) Chemical compound [Fe+2].[O-]S([O-])(=O)=O BAUYGSIQEAFULO-UHFFFAOYSA-L 0.000 description 2
- 229910000359 iron(II) sulfate Inorganic materials 0.000 description 2
- 238000011068 loading method Methods 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 229910052751 metal Inorganic materials 0.000 description 2
- 239000002184 metal Substances 0.000 description 2
- 230000035764 nutrition Effects 0.000 description 2
- 230000036542 oxidative stress Effects 0.000 description 2
- 239000001301 oxygen Substances 0.000 description 2
- 229910052760 oxygen Inorganic materials 0.000 description 2
- 239000013641 positive control Substances 0.000 description 2
- 150000003254 radicals Chemical class 0.000 description 2
- 230000009467 reduction Effects 0.000 description 2
- 230000027756 respiratory electron transport chain Effects 0.000 description 2
- 239000012488 sample solution Substances 0.000 description 2
- 229920006395 saturated elastomer Polymers 0.000 description 2
- 238000001878 scanning electron micrograph Methods 0.000 description 2
- 210000002966 serum Anatomy 0.000 description 2
- 239000012086 standard solution Substances 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- WOAHJDHKFWSLKE-UHFFFAOYSA-N 1,2-benzoquinone Chemical compound O=C1C=CC=CC1=O WOAHJDHKFWSLKE-UHFFFAOYSA-N 0.000 description 1
- NZZDOSANPOZUPK-UHFFFAOYSA-N 4,5-Bisphenol-o-quinone Chemical group C=1C=C(O)C=CC=1C(C)(C)C1=CC(=O)C(=O)C=C1 NZZDOSANPOZUPK-UHFFFAOYSA-N 0.000 description 1
- 208000004998 Abdominal Pain Diseases 0.000 description 1
- 206010010774 Constipation Diseases 0.000 description 1
- 206010012735 Diarrhoea Diseases 0.000 description 1
- 108050000784 Ferritin Proteins 0.000 description 1
- 102000008857 Ferritin Human genes 0.000 description 1
- 238000008416 Ferritin Methods 0.000 description 1
- 102000001554 Hemoglobins Human genes 0.000 description 1
- 108010054147 Hemoglobins Proteins 0.000 description 1
- 241000282412 Homo Species 0.000 description 1
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- 208000002720 Malnutrition Diseases 0.000 description 1
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 239000000853 adhesive Substances 0.000 description 1
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- 150000001335 aliphatic alkanes Chemical group 0.000 description 1
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- 238000013459 approach Methods 0.000 description 1
- 230000003851 biochemical process Effects 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- YCIMNLLNPGFGHC-UHFFFAOYSA-N catechol Chemical group OC1=CC=CC=C1O YCIMNLLNPGFGHC-UHFFFAOYSA-N 0.000 description 1
- 230000024245 cell differentiation Effects 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 230000033077 cellular process Effects 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000000354 decomposition reaction Methods 0.000 description 1
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- 238000010586 diagram Methods 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 210000001198 duodenum Anatomy 0.000 description 1
- 238000005265 energy consumption Methods 0.000 description 1
- 230000009088 enzymatic function Effects 0.000 description 1
- 235000019441 ethanol Nutrition 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 235000013376 functional food Nutrition 0.000 description 1
- 125000000524 functional group Chemical group 0.000 description 1
- 230000001771 impaired effect Effects 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 210000000936 intestine Anatomy 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 150000002506 iron compounds Chemical class 0.000 description 1
- RUTXIHLAWFEWGM-UHFFFAOYSA-H iron(3+) sulfate Chemical compound [Fe+3].[Fe+3].[O-]S([O-])(=O)=O.[O-]S([O-])(=O)=O.[O-]S([O-])(=O)=O RUTXIHLAWFEWGM-UHFFFAOYSA-H 0.000 description 1
- 229910000360 iron(III) sulfate Inorganic materials 0.000 description 1
- 230000001788 irregular Effects 0.000 description 1
- 230000007794 irritation Effects 0.000 description 1
- 210000001630 jejunum Anatomy 0.000 description 1
- 239000003446 ligand Substances 0.000 description 1
- 238000003760 magnetic stirring Methods 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 230000001404 mediated effect Effects 0.000 description 1
- 230000007721 medicinal effect Effects 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 239000002539 nanocarrier Substances 0.000 description 1
- 210000000653 nervous system Anatomy 0.000 description 1
- 230000004770 neurodegeneration Effects 0.000 description 1
- 208000015122 neurodegenerative disease Diseases 0.000 description 1
- 125000004433 nitrogen atom Chemical group N* 0.000 description 1
- 150000002831 nitrogen free-radicals Chemical class 0.000 description 1
- 235000018343 nutrient deficiency Nutrition 0.000 description 1
- 230000010355 oscillation Effects 0.000 description 1
- 230000001590 oxidative effect Effects 0.000 description 1
- 238000003921 particle size analysis Methods 0.000 description 1
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N phenol group Chemical group C1(=CC=CC=C1)O ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 1
- 239000000049 pigment Substances 0.000 description 1
- 230000008092 positive effect Effects 0.000 description 1
- USHAGKDGDHPEEY-UHFFFAOYSA-L potassium persulfate Chemical compound [K+].[K+].[O-]S(=O)(=O)OOS([O-])(=O)=O USHAGKDGDHPEEY-UHFFFAOYSA-L 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 230000008569 process Effects 0.000 description 1
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- 239000000047 product Substances 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 230000005588 protonation Effects 0.000 description 1
- 238000012599 radical scavenging assay Methods 0.000 description 1
- 239000012266 salt solution Substances 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 239000007974 sodium acetate buffer Substances 0.000 description 1
- BHZOKUMUHVTPBX-UHFFFAOYSA-M sodium acetic acid acetate Chemical compound [Na+].CC(O)=O.CC([O-])=O BHZOKUMUHVTPBX-UHFFFAOYSA-M 0.000 description 1
- 238000005507 spraying Methods 0.000 description 1
- 210000002784 stomach Anatomy 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
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- 238000001308 synthesis method Methods 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 239000011573 trace mineral Substances 0.000 description 1
- 235000013619 trace mineral Nutrition 0.000 description 1
- 238000006276 transfer reaction Methods 0.000 description 1
- 235000012431 wafers Nutrition 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
- 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
- A23L33/16—Inorganic salts, minerals or trace elements
- A23L33/165—Complexes or chelates
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
- 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
- A23L33/105—Plant extracts, their artificial duplicates or their derivatives
-
- 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
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
- A61K31/403—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil condensed with carbocyclic rings, e.g. carbazole
- A61K31/404—Indoles, e.g. pindolol
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K33/00—Medicinal preparations containing inorganic active ingredients
- A61K33/24—Heavy metals; Compounds thereof
- A61K33/26—Iron; Compounds thereof
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
- A61P3/02—Nutrients, e.g. vitamins, minerals
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/02—Immunomodulators
- A61P37/04—Immunostimulants
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P39/00—General protective or antinoxious agents
- A61P39/06—Free radical scavengers or antioxidants
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P7/00—Drugs for disorders of the blood or the extracellular fluid
- A61P7/06—Antianaemics
-
- 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
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Veterinary Medicine (AREA)
- Medicinal Chemistry (AREA)
- Public Health (AREA)
- General Health & Medical Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- Pharmacology & Pharmacy (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Immunology (AREA)
- Nutrition Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Epidemiology (AREA)
- Inorganic Chemistry (AREA)
- Polymers & Plastics (AREA)
- Food Science & Technology (AREA)
- Mycology (AREA)
- Diabetes (AREA)
- Hematology (AREA)
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Abstract
The invention discloses a melanin iron with antioxidant activity and a preparation method thereof, wherein the melanin iron is prepared by adopting an iron trichloride method, and the specific steps are as follows: preparing black sesame melanin into BSM solution with a certain concentration, proportionally adding FeCl3.6H2O solution, mixing, regulating pH, optimizing chelation conditions by a response surface method, reacting in a water bath stirring condition with constant temperature and constant speed under dark and normal pressure environment, centrifuging for 5min after chelation reaction is finished, washing precipitate twice, and performing vacuum freeze drying to obtain the ferrous melanin. The melanin iron with antioxidant activity prepared by the preparation method is favorable for human body absorption, has fewer adverse reactions, plays an antioxidant role while treating iron deficiency anemia, and achieves a dual health care function.
Description
Technical Field
The invention relates to the technical field of biology, in particular to ferrous melanin with antioxidant activity and a preparation method thereof.
Background
Iron is a trace element essential to the human body and involved in the necessary biochemical processes such as oxygen transport, electron transfer reactions, gene regulation and cell growth and differentiation. Research shows that iron deficiency not only can cause anemia, but also can increase oxidative stress level, influence immunity and inflammation defense of the organism, and further increase the risk of the organism suffering from cardiovascular diseases, infectious diseases, cancers, diabetes and other diseases. Iron Deficiency Anemia (IDA) is one of the most frequent diseases of nutritional deficiency, and when iron deficiency is severe, it causes problems of digestive tract and nervous system due to impaired intracellular enzyme function, which seriously affects human health. Oral iron supplements are necessary in the treatment and prevention of iron deficiency in humans. Ferrous sulfate has been widely used as an iron supplement for treating iron deficiency anemia, and although it helps to alleviate symptoms of iron deficiency and anemia to some extent, it also causes adverse reactions such as abdominal pain, diarrhea and constipation, and on the other hand, iron intake above the required amount may be toxic and harmful to living beings.
At present, more novel iron supplements are prepared by amino acid or peptide chelated iron, and the iron supplements still have some defects, such as unstable properties, difficult production or storage and easy generation of peculiar smell; the iron supplement has great irritation to the digestive tracts such as the stomach, the intestines and the like, and can cause remarkable increase of gastrointestinal adverse reactions, so the research on the novel iron supplement is urgent to perfect the variety of the iron supplement market in China, and has very important significance for improving the iron nutrition condition of uncomfortable people. Meanwhile, the existing iron-supplementing iron compound has relatively single function, is mainly used for supplementing iron, and has relatively few other matched effects and single purpose.
Black Sesame Melanin (BSM) has various biological activities such as antioxidation, anti-tumor, blood sugar reduction and the like, can provide a plurality of potential binding sites for metal ions, and can form melanin iron (BM-Fe) after reacting with iron ions, and compared with ferrous sulfate, the black sesame melanin can reduce adverse reactions such as gastrointestinal tract irritation and the like caused by free iron ions and has higher bioavailability. Meanwhile, after the iron in the black pigment iron is released, the black sesame melanin can continue to exert other medicinal effects. Therefore, the melanin iron is an ideal medicine which can be used as an iron supplement for treating iron deficiency anemia, can resist oxidization and improve the immunity of organisms.
Disclosure of Invention
The invention aims to prepare the melanin iron with antioxidant activity through the action of the black sesame melanin and the inorganic ferric salt, which is favorable for human body absorption and has less adverse reaction, and the invention can play an antioxidant role while treating iron deficiency anemia, thereby achieving double health care functions.
To achieve the above object, the solution of the present invention is: a preparation method of melanin iron with antioxidant activity adopts an iron trichloride method to prepare the melanin iron, and comprises the following specific steps:
Preparing black sesame melanin into BSM solution with a certain concentration, proportionally adding FeCl 3·6H2 O solution, mixing, regulating pH, optimizing chelation condition by a response surface method, reacting in a dark and normal pressure environment under the conditions of constant temperature and constant speed water bath stirring, centrifuging for 5min after chelation reaction is finished, washing precipitate twice, and vacuum freeze-drying to obtain the melanin iron.
Preferably, the preparation method adopts a response surface method to optimize the chelation condition of the melanin iron, takes the iron chelation rate as an investigation index, takes the volume ratio of the BSM solution to the FeCl 3·6H2 O solution, the reaction time, the reaction temperature, the pH and the rotating speed as investigation factors, sequentially designs a single factor experiment and a response surface experiment, and analyzes the optimal preparation condition.
Preferably, the preparation method is mixed according to the volume ratio of the BSM solution to the FeCl 3·6H2 O solution of 4:4, 5:4, 6:4, 7:4 and 8:4.
Preferably, the reaction time is 1-6 hours, the reaction temperature is 4-60 ℃, the pH is 2-8, and the rotating speed is 0-400rpm.
Preferably, the concentration of the BSM solution is 0.4mg/mL.
Preferably, the FeCl 3·6H2 O solution has a concentration of 0.6mg/mL.
Preferably, the mixture is centrifuged at 8000rpm for 5min.
Aiming at the problems that the iron supplementing chelate in the prior art has single function, the invention prepares the ferrous melanin by reacting with ferric salt solution by utilizing the characteristics of the melanin, can reduce adverse reactions of free iron ions on gastrointestinal tract irritation and the like, has higher bioavailability, and improves the oxidation resistance and the iron content of the iron supplementing agent.
Therefore, the invention also provides the melanin iron with antioxidant activity, which is prepared by the preparation method.
The melanin iron has good oxidation resistance and high iron content, can obviously improve the concentration of hemoglobin and the serum ferritin and serum iron level, and has certain anti-anemia activity.
Therefore, the invention also provides application of the melanin iron in preparing iron supplement products.
After the scheme is adopted, the gain effect of the invention is as follows:
1. the preparation method is simple, the reaction condition is mild, and the preparation method is easy to implement; and the melanin has wide sources and low cost, has excellent physical and chemical properties such as biocompatibility, oxidation resistance, metal chelating property and the like, and is an ideal ligand.
2. The invention adopts a natural melanin-mediated green synthesis method, the iron functionality of the melanin prepared by reacting with iron ions is obviously improved, the oxidation resistance is good, the immunity of the organism can be effectively provided, the iron content is high, the iron is beneficial to the absorption of human bodies, the iron supplement of the human bodies can be promoted, and the double health care function is achieved.
3. The melanin iron prepared by the method disclosed by the invention not only better continues the excellent characteristics of melanin, but also has a better iron supplementing effect, is high in bioavailability and small in side effect, is expected to become a novel nutritional iron supplementing agent, and has a wide development prospect.
Drawings
FIG. 1 is a standard graph of iron content according to the present invention;
FIG. 2 is a graph showing the effect of volume ratio on iron chelation rate according to the present invention;
FIG. 3 is a graph showing the effect of reaction time on iron chelation rate according to the present invention;
FIG. 4 is a graph showing the effect of the reaction temperature on the iron chelation rate according to the present invention;
FIG. 5 is a graph showing the effect of pH on iron chelation rate according to the present invention;
FIG. 6 is a graph showing the effect of the stirring speed on the iron chelation rate according to the present invention;
FIG. 7 is a FTIR diagram of BSM and BM-Fe of the present invention;
FIG. 8 is an SEM image of BSM and BM-Fe of the present invention;
FIG. 9 is a graph of particle size of BSM and BM-Fe of the present invention;
FIG. 10 is a bar graph of BSM, BM-Fe and Vc versus ABTS radical clearance according to the present invention;
FIG. 11 is a bar graph of BSM, BM-Fe and Vc versus DPPH radical scavenging according to the present invention;
FIG. 12 is an iron dissolution pattern of BM-Fe of the present invention in gastrointestinal fluids;
Detailed Description
In order to better understand the present invention, the technical scheme and technical effects of the present invention will be further described below with reference to the accompanying drawings, examples and experimental examples. It should be noted that the following examples are only for explaining the present invention and are not to be construed as limiting the present invention.
The invention provides a preparation method of melanin iron with antioxidant activity, which adopts an iron trichloride method to prepare the melanin iron, and comprises the following specific steps:
Preparing black sesame melanin into BSM solution with the concentration of 0.4mg/mL, mixing the BSM solution with FeCl 3·6H2 O solution with the concentration of 0.6mg/mL according to a certain proportion, regulating pH, optimizing the chelation condition by a response surface method, reacting for a period of time under the conditions of constant temperature and constant speed water bath stirring in dark and normal pressure environment, centrifuging for 5min at the rotating speed of a centrifugal machine of 8000rpm after the chelation reaction is finished, washing the precipitate twice, and performing vacuum freeze drying to obtain the ferrous melanin. In this embodiment, a response surface experiment is designed to examine the influence of the volume ratio of the BSM solution to the FeCl 3·6H2 O solution, the reaction time, the reaction temperature, the pH, and the rotation speed on the chelation reaction, and the optimal process conditions for optimizing the melanin iron are as follows: the volume ratio of the BSM solution to the FeCl 3·6H2 O solution is 4: 4. the pH value is 3, and the iron chelation rate reaches the maximum value when the reaction is carried out for 4 hours under the water bath stirring condition that the reaction temperature is 50 ℃ and the rotating speed is 300rpm under the dark and normal pressure environment.
Determination of chelation Rate: by o-phenanthroline method
Drawing a standard curve: accurately preparing 20ug/mL of iron ion standard solution by using ferric sulfate [ FeNH 4(SO4)2·12H2 O ], diluting for 10 times to obtain an iron standard test solution, accurately measuring 2.0, 4.0, 6.0, 8.0, 10.0, 12.0 and 14.0mL of the iron standard test solution, respectively placing the iron standard test solutions into 50mL volumetric flasks, adding 1.0mL of 10% hydroxylamine hydrochloride, 5.0mL of acetic acid-sodium acetate buffer solution and 3.0mL of 0.1% phenanthroline, diluting to scale by using distilled water, and shaking uniformly. Taking a standard solution without adding iron as a blank control, measuring the absorbance at a wavelength of 510nm by using spectrophotometric juice, drawing a standard curve, and fig. 1 is a standard curve chart of the iron content of the invention, wherein the regression equation is as follows:
Y＝0.0073X+0.0024R2＝0.9998
Determination of iron content: and precisely measuring the sample test solution in a volumetric flask with a volume of 5.0mL to 50mL, drawing a standard curve of the step, and substituting the standard curve into a regression equation to calculate the content of the standard curve.
Iron content/(mg/kg) =c/[ m (V 1/V0) ]
Wherein: c-checking the corresponding iron content of the chelate solution in mug on a standard curve; m-chelate mass, g; v 1 -measuring the volume of the chelate solution, mL; v 0 -fixed volume after chelate treatment, mL.
Determination of iron chelation Rate
Chelation rate/% = [ (C 0-Ce)/C0 ] ×100%
Wherein: c 0 -initial concentration of Fe 3+ ions in solution, mg/L; equilibrium concentration of ions in Ce-post-reaction solution, mg/L
Example 1
The influence of 5 factors, namely the volume ratio, the reaction time, the reaction temperature, the pH value and the rotating speed, on the iron chelation rate is examined by adopting a single-factor experiment, and the specific design is shown in table 1.
Table 1 single factor experimental design table
A study of the Effect of the volume ratio of BSM solution to FeCl 3·6H2 O solution on the iron chelation Rate
Under the conditions of reaction time of 5h, reaction temperature of 30 ℃, pH value of 5 and rotating speed of 200rpm, mixing BSM solution and FeCl 3·6H2 O solution according to volume ratio of 4:4, 5:4, 6:4, 7:4 and 8:4 to prepare five groups of melanin iron, taking iron chelation rate as investigation index, investigating the influence of different volume ratios on BM-Fe iron chelation rate, and FIG. 2 is a graph of the influence of the volume ratio on iron chelation rate.
As can be seen from fig. 2, as the proportion of the BSM solution increases, the iron chelation rate generally tends to increase and then decrease. When the volume ratio of the BSM solution to the FeCl 3·6H2 O solution is 4:4-6:4, the iron chelation rate increases with the increase of the BSM, but the increase is not obvious; when the ratio is 7:4-8:4, the iron chelation rate is obviously reduced along with the increase of the BSM, because the volume ratio is too small, the BSM is insufficient, a stable structure cannot be formed, and the chelate is unstable; when the volume ratio is too large, the content of BSM is too high, and the chelation of BSM to Fe 3+ is saturated, so that the utilization rate of BSM is reduced, the iron chelation rate is reduced, and the BSM is wasted. When the volume ratio is 6:4, the iron chelation rate reaches the maximum value and is obviously different from other conditions, so that the optimal volume ratio is 6:4
B study of the Effect of reaction time on iron chelation Rate
Mixing BSM solution and FeCl 3·6H2 O solution according to volume ratio of 6:4, controlling reaction time to be 1,2,3,4, 5 and 6 hours under the conditions of pH value of 5, reaction temperature of 30 ℃ and rotating speed of 200rpm, preparing six groups of melanin iron, taking iron chelating rate as investigation index, and investigating influence of different reaction time on BM-Fe iron chelating rate.
As can be taken from fig. 3, overall, the iron chelation rate increases all the time from 1 to 6 hours, significantly increasing at 1 to 2 hours, with the subsequent increase not being significant and the range of variation not being large. This is because the chelation reaction is a relatively rapid reaction, and if the time is too short, the chelation reaction is not complete, the chelation rate is low, and if the time is too long, the chelation of the BSM to the Fe 3+ is saturated, so the reaction time is preferably selected to be 5 hours.
C, researching influence of reaction temperature on iron chelation rate
Mixing BSM solution and FeCl 3·6H2 O solution according to volume ratio of 6:4, controlling reaction temperature to be 4, 30, 40, 50 and 60 ℃ under the conditions of pH value of 5, rotating speed of 200rpm and reaction time of 5 hours, preparing five groups of melanin iron, taking iron chelation rate as investigation index, and investigating influence of different reaction temperatures on BM-Fe iron chelation rate.
As can be seen from fig. 4, the iron chelation rate increases and then decreases with increasing temperature. At 4-30 ℃, the iron chelation rate is obviously increased along with the temperature rise; after exceeding 40 ℃, the iron chelation rate decreases significantly as the temperature continues to rise. This is because the reaction cannot be completely performed due to the excessively low temperature; too high a temperature, while accelerating the reaction rate, greatly increases the energy consumption, adversely affecting the formation of complexes, and the elevated temperature may cause oxidation of BSM, decomposition of complexes, or other reactions to occur, thereby reducing the iron chelating rate. Thus, the optimal temperature for the chelation reaction is 30 ℃.
D study of the influence of pH on iron chelation Rate
Mixing BSM solution and FeCl 3·6H2 O solution according to volume ratio of 6:4, regulating pH value to 2,3, 4, 5, 6, 7 and 8 under the conditions of reaction temperature of 30 ℃, rotating speed of 200rpm and reaction time of 5h, preparing seven groups of melanin iron, taking iron chelating rate as investigation index, and investigating the influence of different pH values on BM-Fe iron chelating rate.
FIG. 5 shows that at pH 2, the iron chelation rate is only 5%, probably because a large amount of hydrated hydrogen ions in the solution compete with metal ions for reactive sites, and probably because the solution is strongly acidic, phenolic hydroxyl groups, carboxyl groups, amine groups and the like in BSM molecules are positively charged due to protonation, and stronger electrostatic repulsion is generated between the solution and Fe 3+, so that Fe 3+ is difficult to approach the BSM, and the chelation rate of Fe 3+ is almost zero; as the pH value increases, the chelation rate increases because the acidity of the solution is weakened, and the active sites in the adsorbent, which are complexed with hydronium ions, are gradually exposed, so that chelation of Fe 3+ is facilitated; the overall tendency is that the pH value is lower after being higher than 4, probably because the upper limit of iron ion microprecipitation is exceeded, and metal ions can form oxide precipitates; after the pH is greater than 8, OH < - > in the solution competes with BSM for Fe 3+, so that the concentration of free Fe 3+ in the solution becomes small, and the chelation rate is reduced, and therefore, no further investigation is continued. Acidity thus has a major effect on BSM chelating Fe 3+, with an optimum pH of 3.
E study of the Effect of rotational speed on iron chelation Rate
Mixing a BSM solution and a FeCl 3·6H2 O solution according to a volume ratio of 6:4, controlling the rotating speed to be 0, 100, 200, 300 and 400rpm under the conditions of pH value of 5, reaction temperature of 30 ℃ and reaction time of 5 hours, preparing five groups of melanin iron, taking the iron chelating rate as an investigation index, and investigating the influence of different rotating speeds on BM-Fe iron chelating rate.
As can be seen from fig. 6, whether or not the stirring has a significant influence on the iron chelation rate, the iron chelation rate increases significantly after the magnetic stirring, but the chelation rate is maximum in the test range at a rotation speed of 300rpm. Therefore, the optimum rotation speed is 300rpm.
Example 2
Based on a single factor experiment, a response surface method is used for optimizing the preparation process. And (3) taking the chelation rate (%) of BM-Fe as a response value, and carrying out Box-Behnken Design and response surface analysis experiments by using Design Expert 10 statistical software. Three levels were respectively designed for experiments by selecting four factors of the volume ratio X 1, the reaction time X 2 (h) and the reaction temperature X 3(℃)、PH X4, and the specific results are shown in Table 2.
TABLE 2 response surface Experimental design Table
According to the experimental result of the response surface, performing multiple quadratic regression fitting on the relationship between the black sesame melanin-nano iron chelation rate Y and the corresponding volume ratio X1, the reaction time X2 (h), the reaction temperature X3 (DEG C) and the pH value X4 by using Design Expert 10 software to obtain a model of a multiple regression equation, namely:
Y＝77.56-7.42X1-1.43X2+2.42X3-3.86X4-1.57X1X2-2.89X1X3-3.64X1X4-0.28X2X3+1.81X2X4-1.54X3X4+1.42X1 2+2.93X2 2+1.48X3 2-1.53X4 2
As can be seen from Table 5, when the chelation rate of BM-Fe is used as a response value, the P value of the equation model is less than 0.05, which shows that the quadratic regression equation model is obvious and has statistical significance; meanwhile, the P value of the mismatching term is more than 0.05, which shows that the mismatching term of the equation does not obviously indicate that the fitting condition of the equation to the experiment is good, the mismatching factor is not present, the experimental error is small, and the model is successfully established. According to the P value, the primary and secondary orders of factors affecting the BM-Fe chelation rate are as follows: volume ratio (X 1)＞pH(X4) > reaction temperature (X 3) > reaction time (X 2); the P values of interaction terms X1X2、X1X3、X1X4、X2X3、X2X4、X3X4 are all greater than 0.05, indicating that the interaction term effect is insignificant, indicating that the four factors have no interaction. The optimal theoretical preparation process parameters of BM-Fe are obtained through software analysis: the volume ratio is 3.988:4, the reaction time is 4 hours, the reaction temperature is 50 ℃, and the pH is 3; in view of convenience and feasibility of practical operation, the extraction process conditions are adjusted as follows: the volume ratio is 4:4, the reaction time is 4h, the reaction temperature is 50 ℃, and the pH is 3.
TABLE 5 regression equation analysis of variance results
Experimental example 1
In this experimental example, infrared spectrum experiments were performed on BM-Fe and BSM prepared under the optimal conditions obtained in examples 1-2, and before the experiments, BSM and BM-Fe were respectively ground in an agate bowl with potassium bromide (KBr), then wafers were prepared, and infrared spectra were obtained by measurement with a Fourier infrared spectrometer (FT-IR) and compared.
As a result, as shown in FIG. 7, the absorption peaks of BSM were mainly located on several groups of absorption peaks of 3365cm -1、2930cm-1、1625cm-1、1425cm-1; the absorption peak at 3365cm -1 is very broad and strong, which indicates that the BSM contains hydroxyl (-OH) and amino (-NH 2) and the region also belongs to the telescopic vibration of carbonyl (-C=O-) and the small and sharp absorption peak at 2930cm -1 is caused by the telescopic vibration of C-H in an alkane structure, and the strong absorption peak at 1625cm -1、1425cm-1 is caused by the vibration of an aromatic ring and is the vibration telescopic absorption peak of the aromatic ring C=C; the strong absorption peak at 1625cm -1 indicates the existence of carbonyl in the molecule, and the strong absorption peak at 3400cm -1 indicates the existence of-COOH structure, the range of 810-525cm -1 is aromatic ring substituted region, the weak absorption peak indicates that aromatic ring is substituted, the aromatic hydrogen content is less, and a conjugated system is formed. Compared with BSM, the infrared absorption peak of 3400-3000cm -1、1500cm-1 and fingerprint area in BM-Fe confirms the existence of BSM, and the partial absorption peak is weakened, which indicates that carboxyl, hydroxyl and amino are involved in the chelation of metal ions. The absorption peak at 3365cm -1 is weakened, which means that the strength of-OH or amino is reduced, the N atom of amino may undergo coordination reaction and even disappear, and the stretching vibration frequency of-N-H group is reduced, which means that the chemical combination of iron ions and BSM is reduced, several characteristic peak changes can reflect that the reaction of BSM and ferric trichloride hexahydrate has no great influence on the overall structure of BSM, but from the peak change of some characteristic absorption peaks, fe 3+ is mainly complexed with amino, carboxyl and phenolic hydroxyl in BSM.
Experimental example 2
The experimental example is to observe the surfaces of BM-Fe and BSM prepared under the optimal conditions obtained in the examples 1-2, and the BSM and BM-Fe are placed on a sample stage by using double-sided conductive adhesive before the experiment, and then subjected to metal spraying treatment, and the appearance is observed under a scanning electron microscope. FIG. 8 is an SEM image of BSM and BM-Fe, wherein A, C is BSM and B, D is BM-Fe.
From fig. 8, it can be seen that the two microscopic morphologies have a significant difference, the BSM has a cubic block structure, the structure is more obvious, the surface is free from attachments, and the morphology is more regular. BM-Fe is more aggregated, flaky, and has a mostly irregular shape and a rough surface, and the result is probably due to the fact that some groups of the BM-Fe can be changed after BSM reacts with iron, and the internal structure is changed, so that the surface morphology of BM-Fe is changed.
Experimental example 3
In this experimental example, the BM-Fe and BSM prepared under the optimal conditions obtained in examples 1-2 were subjected to dynamic light scattering particle size analysis, and the prepared BSM and BM-Fe were diluted by the same factor and the particle size was measured by a dynamic light scattering instrument. FIG. 9 is a graph of particle sizes of BSM and BM-Fe according to the present invention, wherein A is BSM and B is BM-Fe.
As shown in FIG. 9, the data of the average particle diameters thereof were 844.9nm and 294.3nm, respectively. The particle size of BM-Fe is significantly smaller than BSM, probably because the iron ions are combined with carboxyl and hydroxyl groups on BSM to form nano-carriers of the iron ions, resulting in reduced particle size of BM-Fe.
Experimental example 4
This experimental example conducted oxidation resistance test on BM-Fe prepared under the optimal conditions obtained in examples 1-2.
(1) ABTS free radical scavenging assay
The ABTS ionic liquid is prepared by mixing 7mM of ABTS and 2.45mM of potassium persulfate solution in the dark for reaction for 12-16 hours, and the absorbance value of the ABTS ionic liquid at 734nm is adjusted to be 0.7+/-0.02 by ethanol before use. The reagents in the reaction are tested according to a sample adding sequence table, and the details are shown in a table 3, wherein BSM and BM-Fe solutions are respectively diluted into 5 concentrations (1.25, 2.5, 5, 10 and 20 mug/mL) in turn, the reaction is carried out for 6min at room temperature in a light-shielding vortex oscillation mode, and the absorbance value is measured at 734 nm. The positive control was Vc at the same concentration, and the scavenging ability of the ABTS free radical was calculated according to the following formula:
wherein: a 1 is the absorbance value of the sample after reaction with the ABTS free radical; a 2 is the absorbance value of the absolute ethyl alcohol of the control group after the absolute ethyl alcohol reacts with the ABTS free radical; a 0 is the initial absorbance value of the ABTS radical solution.
TABLE 3 sample loading sequence table
As can be seen from FIG. 10, the inhibition ratios of BSM, BM-Fe, vc to ABTS all showed a certain concentration dependence. The cleaning efficiency of BSM and BM-Fe is obviously higher than that of Vc group. The BSM molecule has an oxidizing (o-quinone) and a reducing (o-hydroquinone) group, and can remove active oxygen or active nitrogen radicals by losing electrons or capturing electrons during electron transfer through interaction with the radicals. In addition, the functional groups with different oxidation resistance can be reduced to generate synergistic effect, so that the oxidation resistance of the BSM is stronger. And the clearance effect of BM-Fe is equivalent to that of BSM, which shows that the anti-oxidation activity of BSM is better maintained after the BSM reacts with ferric trichloride.
(2) DPPH radical scavenging test
Accurately weighing 25.6mg of DPPH free radical, shaking uniformly in a 100mL volumetric flask with absolute ethyl alcohol to constant volume, and preserving in a dark place for later use. In the reaction, each reagent is tested according to a sample addition sequence table, wherein BSM and BM-Fe solutions are respectively diluted into 5 concentrations (12.5, 25, 50, 100 and 200 mu g/mL) in turn, VC solutions with the same concentration are used as positive controls, shading reaction is carried out for 30min at room temperature, and absorbance value A is measured at 517nm wavelength. The clearance of DPPH radicals was calculated as follows:
TABLE 4 sample loading sequence table
As can be seen from FIG. 11, the inhibition ratios of BSM and BM-Fe to DPPH all showed a certain concentration dependence. But the cleaning efficiency of BSM and BM-Fe is significantly lower than that of Vc group. When the concentration is 200 mug/mL, the clearance rate can reach about 40%, and the antioxidation effect is better. BSM and its derivatives may directly capture or bind to DPPH to scavenge DPPH radicals several derivatives of BSM have a lower clearance than BSM, probably because of the electron loss due to the conversion of the bisphenol/benzoquinone structure when amino acids and BSM act, reducing their free radical scavenging activity. And BM-Fe scavenging efficiency is comparable to BSM.
In general, iron-containing enzymes, which play an important role in regulating various cellular processes in the body, are reduced in activity due to iron deficiency, and the reduction of the activity of these enzymes seriously affects the normal metabolism of the body, resulting in an increase in oxidation level and an increase in oxidative stress, thereby leading to the occurrence of cardiovascular diseases, infectious diseases, cancers, diabetes, neurodegenerative diseases, and the like. The comprehensive analysis of the scavenging effect of BSM and BM-Fe on ABTS and DPPH free radicals can find that BM-Fe better continues the strong antioxidation of BSM, and BM-Fe is used as a potential novel iron supplement agent and may have positive therapeutic effect on related diseases caused by anemia.
Experimental example 5
In vitro iron dissolution experiments were performed on BM-Fe prepared under the optimal conditions obtained in examples 1-2.
(1) Simulated gastric fluid
Accurately weighing 20mg of BM-Fe, dissolving the BM-Fe in 100mL of hydrochloric acid solution (0.1 moL/L), magnetically stirring the solution at 37 ℃ and 100rpm for 2 hours, taking a sample every 0.5 hour, taking 3mL of the sample, and simultaneously adding hydrochloric acid solution with the same volume. After the reaction, the sample solution is filtered by a microporous filter membrane with the thickness of 0.45 mu m, 2mL of each filtrate is respectively taken out in a volumetric flask with the thickness of 50mL after the filtration, and then the operation steps such as iron content measurement are carried out, and the absorbance value is measured at the position of 510nm after the completion.
As can be seen from FIG. 12, the release rate of BM-Fe was about 45% after 30min in the gastric juice environment, which is probably due to the strong acid condition of gastric juice increasing the free iron ion content released by BM-Fe. At 2h, the iron release rate of BM-Fe exceeds 80%.
(2) Simulated intestinal juice
Accurately weighing 20mg of BM-Fe, dissolving the BM-Fe in 100mL of buffer solution with pH of 6.8, magnetically stirring the solution at 37 ℃ and 100rpm for reaction for 4 hours, taking a sample every 0.5 hour, taking 3mL at a time, and simultaneously adding the buffer solution with the same dosage. After the reaction, the sample solution is filtered by a microporous filter membrane with the thickness of 0.45 mu m, 2mL of each filtrate is respectively taken out in a volumetric flask with the thickness of 50mL after the filtration, and then the operation steps such as iron content measurement are carried out, and the absorbance value is measured at the position of 510nm after the completion.
As shown in FIG. 12, BM-Fe has a high release rate in intestinal juice environment, and the release rate of BM-Fe reaches 70% at 2 hours, because iron ions are mainly absorbed in the duodenum and upper jejunum, the iron in BM-Fe has better bioavailability. All experiments in this case were repeated 3 times and the results are expressed as mean X.+ -. SD. Graphical drawing was performed using Origin 2018, SPSS 24.0 for significance analysis, with different lower case letters indicating significant differences (P < 0.05).
As can be seen from the above experimental examples 1 to 5, elemental iron successfully reacted with BSM; the BM-Fe has higher iron element dissolution in the simulated gastrointestinal environment, which shows that the BM-Fe has good bioavailability in the gastrointestinal environment and better retains the excellent characteristics of melanin such as antioxidation, etc.
Wherein, the infrared result shows that carboxyl, hydroxyl and amino groups of the BSM participate in the chelation of iron ions; the oxidation resistance experiment shows that BM-Fe better extends the excellent oxidation resistance of BSM; the in vitro iron dissolution experimental result shows that BM-Fe is favorable for organism absorption.
The existing iron supplementing chelate has been proved to have good blood supplementing effect, but has no attention in the aspects of antioxidation and the like, and the research proves that the melanin iron has good antioxidation, good human body absorbability and is beneficial to human body utilization. Therefore, the BM-Fe complex is expected to be developed into a novel multifunctional iron supplement with good activity, and has great prospect in application of novel functional foods with potential positive effects on health in biology, medicine and even basic nutrition.
The above embodiments are only preferred embodiments of the present invention, and are not limited to the present invention, and all equivalent changes made according to the design key of the present invention fall within the protection scope of the present invention.
Claims (2)
1. A preparation method of melanin iron with antioxidant activity is characterized by adopting an iron trichloride method to prepare the melanin iron, and comprises the following specific steps:
Preparing black sesame melanin into BSM solution with the concentration of 0.4mg/mL, proportionally adding FeCl 3·6H2 O solution with the concentration of 0.6mg/mL, mixing, adjusting pH, optimizing chelation conditions by a response surface method, reacting in a dark and normal pressure environment under the conditions of constant temperature and constant speed water bath stirring, centrifuging for 5min under the condition that the rotating speed of a centrifugal machine is 8000rpm after chelation reaction is finished, washing precipitate twice, and performing vacuum freeze drying to obtain ferrous melanin;
The preparation method adopts a response surface method to optimize the chelation condition of the ferrous melanin, takes the iron chelation rate as an investigation index, takes the volume ratio of a BSM solution to a FeCl 3·6H2 O solution, the reaction time, the reaction temperature, the pH and the rotating speed as investigation factors, sequentially designs a single factor experiment and a response surface experiment, and analyzes the optimal preparation conditions as follows: the volume ratio of the BSM solution to the FeCl 3·6H2 O solution is 4: 4. the pH is 3, and the reaction is carried out for 4 hours under the water bath stirring condition that the reaction temperature is 50 ℃ and the rotating speed is 300rpm in dark and normal pressure environment.
2. An iron melanin having antioxidant activity, which is prepared by the method for preparing an iron melanin having antioxidant activity as claimed in claim 1.
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