CN113875990A - Ferrous iron with antioxidant activity and preparation method thereof - Google Patents
Ferrous iron with antioxidant activity and preparation method thereof Download PDFInfo
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- CN113875990A CN113875990A CN202111179686.1A CN202111179686A CN113875990A CN 113875990 A CN113875990 A CN 113875990A CN 202111179686 A CN202111179686 A CN 202111179686A CN 113875990 A CN113875990 A CN 113875990A
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- iron
- melanin
- bsm
- solution
- antioxidant activity
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- 230000003078 antioxidant effect Effects 0.000 title claims abstract description 17
- 238000002360 preparation method Methods 0.000 title claims abstract description 16
- CWYNVVGOOAEACU-UHFFFAOYSA-N Fe2+ Chemical compound [Fe+2] CWYNVVGOOAEACU-UHFFFAOYSA-N 0.000 title claims abstract description 11
- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 claims abstract description 251
- 229910052742 iron Inorganic materials 0.000 claims abstract description 129
- 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 claims abstract description 82
- 238000006243 chemical reaction Methods 0.000 claims abstract description 46
- 238000000034 method Methods 0.000 claims abstract description 24
- RBTARNINKXHZNM-UHFFFAOYSA-K iron trichloride Chemical compound Cl[Fe](Cl)Cl RBTARNINKXHZNM-UHFFFAOYSA-K 0.000 claims abstract description 20
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- 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
- A23L33/16—Inorganic salts, minerals or trace elements
- A23L33/165—Complexes or chelates
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- 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
- A23L33/105—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
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- 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
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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- 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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- A61P7/06—Antianaemics
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- 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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Abstract
The invention discloses a ferrous iron with antioxidant activity and a preparation method thereof, wherein a ferric trichloride method is adopted to prepare the ferrous iron, and the preparation method comprises the following specific steps: preparing black sesame melanin into BSM solution with a certain concentration, adding FeCl3 & 6H2O solution in proportion, mixing, adjusting pH, optimizing chelation conditions by a response surface method, reacting under constant-temperature and constant-speed water bath stirring conditions in a dark and normal-pressure environment, centrifuging for 5min after the chelation reaction is finished, taking precipitate, washing twice, and carrying out vacuum freeze drying to obtain the melanin iron. The melanin iron with antioxidant activity prepared by the preparation method disclosed by the invention is beneficial to absorption by a human body and has fewer adverse reactions, and the melanin iron can play an antioxidant role while treating iron-deficiency anemia, so that double health-care functions are achieved.
Description
Technical Field
The invention relates to the technical field of biology, and particularly relates to a ferrous oxide with antioxidant activity and a preparation method thereof.
Background
Iron is a trace element essential to the human body and is involved in essential biochemical processes such as oxygen transport, electron transfer reactions, gene regulation, and cell growth and differentiation. Research shows that iron deficiency not only causes anemia, but also increases oxidative stress level, influences body immunity and inflammation defense, and further increases the risk of cardiovascular diseases, infectious diseases, cancers, diabetes and other diseases. Iron Deficiency Anemia (IDA) is one of the diseases with the highest incidence of nutrient deficiency, and when iron deficiency is serious, the function of intracellular enzymes is damaged, so that problems of the digestive tract and the nervous system are generated, and the health of human beings is seriously influenced. 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 the treatment of iron deficiency anemia, and although it is helpful to some extent to relieve the symptoms of iron deficiency and anemia, it also causes adverse reactions such as epigastric pain, diarrhea and constipation, and on the other hand, it may be toxic and harmful to living beings when the iron intake is higher than necessary.
At present, the novel iron supplement is mostly prepared by amino acid or peptide chelated iron, and the iron supplement still has some defects, such as unstable properties, difficult production or storage and easy generation of peculiar smell; the iron supplement has large irritation to digestive tracts such as stomach, intestine and the like, and adverse reactions of gastrointestinal tracts are possibly increased remarkably, so that the research on novel iron supplements to perfect the variety of iron supplement markets in China is imminent, and the iron supplement has very important significance for improving the iron nutrition condition of uncomfortable people. Meanwhile, the existing iron and iron supplement compound has relatively single function and is mainly used for iron supplement, and meanwhile, the compound has relatively few other matched effects and single use.
The Black Sesame Melanin (BSM) has multiple biological activities of oxidation resistance, tumor resistance, blood sugar reduction and the like, can provide multiple potential binding sites for metal ions, forms melanin iron (BM-Fe) after reacting with iron ions, and can reduce adverse reactions such as gastrointestinal irritation and the like caused by free iron ions and has higher bioavailability compared with ferrous sulfate. Meanwhile, after the iron in the melanin iron is released, the black sesame melanin can continuously exert other drug effects. Therefore, the melanin iron is an ideal medicine which can be used as an iron supplement to treat iron-deficiency anemia, resist oxidation and improve the immunity of the organism.
Disclosure of Invention
The invention aims to prepare the melanin iron with antioxidant activity by the action of the black sesame melanin and inorganic iron salt, is beneficial to human body absorption and has less adverse reaction, and the melanin iron can play the role of antioxidation while treating iron-deficiency anemia to achieve double health-care functions.
In order to achieve the above purpose, the solution of the invention is: a method for preparing melanin iron with antioxidant activity adopts a ferric chloride method to prepare the melanin iron, and comprises the following specific steps:
preparing black sesame melanin into BSM solution with a certain concentration, and adding FeCl according to a certain proportion3·6H2Mixing O solutions, adjusting pH, optimizing chelating conditions by a response surface method, reacting under the conditions of constant temperature and constant speed water bath stirring in a dark and normal pressure environment, centrifuging for 5min after the chelating reaction is finished, taking a precipitate, washing twice, and performing vacuum freeze drying to obtain the ferrous black.
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, and takes the BSM solution and FeCl3·6H2The volume ratio, the reaction time, the reaction temperature, the pH value and the rotating speed of the O solution are considered factors, and a single-factor experiment is designed in sequenceAnd response surface experiments, analyzing the optimal preparation conditions.
Preferably, the preparation method comprises the steps of mixing BSM solution with FeCl3·6H2The volume ratio of the O solution is 4:4, 5:4, 6:4, 7:4 and 8: 4.
Preferably, the reaction time is 1-6h, the reaction temperature is 4-60 ℃, the pH is 2-8, and the rotation speed is 0-400 rpm.
Preferably, the concentration of the BSM solution is 0.4 mg/mL.
Preferably, the FeCl3·6H2The concentration of the O solution was 0.6 mg/mL.
Preferably, the mixed liquid is centrifuged for 5min at 8000rpm of the centrifuge.
Aiming at the single function of the iron supplement chelate in the prior art, the invention prepares the ferrous iron by utilizing the characteristics of melanin and reacting with an iron salt solution, can reduce adverse reactions such as stimulation of free iron ions to gastrointestinal tracts and the like, has higher bioavailability, and improves the oxidation resistance and the iron content of the iron supplement.
Therefore, the invention also provides the ferrous oxide with the antioxidant activity, which is prepared by the preparation method.
The ferrous iron provided by the invention has good oxidation resistance and high iron content, can obviously improve the hemoglobin concentration and the levels of serum ferritin and serum iron, and has certain anti-anemia activity.
Therefore, the invention also provides the 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 implementation is easy; and the melanin has wide sources, low cost, excellent physical and chemical properties such as biocompatibility, oxidation resistance, metal chelation and the like, and is an ideal ligand.
2. The invention adopts a natural melanin-mediated green synthesis method, the functionality of the prepared melanin iron is obviously improved after the melanin iron reacts with iron ions, the oxidation resistance is good, the immunity of the organism can be effectively provided, the iron content is high, the absorption of the human body is facilitated, the supplement of the iron of the human body can be promoted, and the double health-care functions are achieved.
3. The melanin iron prepared by the method not only has good characteristics of melanin, but also has good iron supplementing effect, high bioavailability and small side effect, is expected to become a novel nutritional iron supplement, and has wide development prospect.
Drawings
FIG. 1 is a graph of a standard iron content curve 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 reaction temperature on 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 stirring speed on iron chelation rate according to the present invention;
FIG. 7 is a FTIR plot of BSM and BM-Fe of the present invention;
FIG. 8 is an SEM image of a BSM and BM-Fe of the present invention;
FIG. 9 is a graph of the 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 of the present invention;
FIG. 11 is a bar graph of BSM, BM-Fe and Vc versus DPPH radical clearance in accordance with the present invention;
FIG. 12 is an iron elution diagram of BM-Fe of the present invention in gastrointestinal fluids;
Detailed Description
For better understanding of the present invention, the technical solutions and 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 illustrating 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 a ferric trichloride method to prepare the melanin iron and comprises the following specific steps:
black will beThe sesame melanin is prepared into BSM solution with the concentration of 0.4mg/mL and FeCl with the concentration of 0.6mg/mL3·6H2Mixing the O solution according to a certain proportion, adjusting pH, optimizing chelation conditions by a response surface method, reacting for a period of time under the conditions of constant temperature and constant speed water bath stirring in the dark and normal pressure environment, centrifuging for 5min under the condition that the rotating speed of a centrifugal machine is 8000rpm after the chelation reaction is finished, taking precipitate, washing twice, and carrying out vacuum freeze drying to obtain the ferrous ferrite. In this example, a response surface experiment was designed to examine BSM solution and FeCl3·6H2The influence of the volume ratio, the reaction time, the reaction temperature, the pH value and the rotating speed of the O solution on the chelation reaction is optimized, and the optimal process conditions for optimizing the melanin iron are as follows: BSM solution and FeCl3·6H2The volume ratio of the 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 conditions of water bath stirring at the reaction temperature of 50 ℃ and the rotating speed of 300rpm in the dark and normal pressure environment.
Determination of chelation rate: by the o-diazepine process
Drawing a standard curve: with ferric ammonium sulphate [ FeNH ]4(SO4)2·12H2O]Precisely preparing 20ug/mL of iron ion standard solution, diluting by 10 times to obtain iron standard test solution, precisely measuring 2.0, 4.0, 6.0, 8.0, 10.0, 12.0 and 14.0mL of iron standard test solution, respectively placing 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 with distilled water to scale, and shaking. Taking a standard solution without iron as a blank control, measuring the absorbance at the wavelength of 510nm by using spectrophotometric juice, and drawing a standard curve, wherein FIG. 1 is a standard curve graph of the iron content of the invention, and the regression equation is as follows:
Y＝0.0073X+0.0024R2＝0.9998
and (3) determination of iron content: precisely measuring a sample test solution in a volumetric flask from 5.0mL to 50mL, drawing a standard curve in the step, and substituting the standard curve into a regression equation to calculate the content of the sample test solution.
Iron content/(mg/kg) ═ C/[ m (V)1/V0)]
In the formula: checking the corresponding iron content of the chelate solution, namely mu g, on a C-standard curve; m-chelateMass of compound, g; v1The volume of chelate solution taken, mL, at the time of determination; v0Volume fixed after chelate treatment, mL.
Determination of iron chelation Rate
Chelation rate/% ([ (C)0-Ce)/C0]*100％
In the formula: c0Fe in solution3+Initial concentration of ions, mg/L; equilibrium concentration of ions in the solution after Ce-reaction, mg/L
Example 1
The influence of 5 factors of volume ratio, reaction time, reaction temperature, pH value and 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 experiment design Table
Study of BSM solution and FeCl3·6H2Influence of volume ratio of O solution on iron chelation ratio
Under the conditions of reaction time of 5h, reaction temperature of 30 ℃, pH value of 5 and rotation speed of 200rpm, BSM solution and FeCl3·6H2Mixing O solution according to the volume ratio of 4:4, 5:4, 6:4, 7:4 and 8:4 to prepare five groups of melanin iron, and observing the influence of different volume ratios on the iron chelation rate of BM-Fe by taking the iron chelation rate as an observation index, wherein FIG. 2 is a result graph of the influence of the volume ratio on the iron chelation rate of the invention.
As can be seen from fig. 2, as the BSM solution ratio increases, the iron chelation rate generally tends to increase first and then decrease. BSM solution and FeCl3·6H2When the volume ratio of the O solution is 4:4-6:4, the iron chelation rate is increased along with the increase of BSM, but the increase is not obvious; when the ratio is 7:4-8:4, the iron chelation rate is remarkably reduced along with the increase of 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 BSM is excessive to Fe3+The chelation of (b) is already saturated, so that the BSM utilization rate decreases, the iron chelation rate decreases, and BSM waste may occur. WhileThe iron chelation rate reached a maximum at a volume ratio of 6:4, and was significantly different from other conditions, so the optimum volume ratio was selected to be 6:4
B study of the Effect of reaction time on iron chelation Rate
Mixing BSM solution with FeCl3·6H2Mixing O solutions according to a volume ratio of 6:4, controlling reaction time to be 1, 2, 3, 4, 5 and 6h under the conditions that the pH value is 5, the reaction temperature is 30 ℃ and the rotating speed is 200rpm, preparing six groups of ferrous iron, and observing the influence of different reaction times on the BM-Fe iron chelation rate by taking the iron chelation rate as an observation index.
As can be taken from fig. 3, overall, the iron chelation rate increased all the time from 1 to 6h, increased significantly at 1 to 2h, and then increased insignificantly, with no wide variation. This is because the chelation reaction is a faster reaction, and if the time is too short, the chelation reaction is incomplete, the chelation rate is low, and if the time is too long, BSM is responsible for Fe3+The chelation of (2) is already saturated, so that a reaction time of 5h is preferably chosen.
C study of the influence of reaction temperature on iron chelation Rate
Mixing BSM solution with FeCl3·6H2Mixing O solutions according to a volume ratio of 6:4, controlling reaction temperatures of 4, 30, 40, 50 and 60 ℃ under the conditions of pH value of 5, rotation speed of 200rpm and reaction time of 5h to prepare five groups of melanin iron, and observing the influence of different reaction temperatures on the BM-Fe iron chelation rate by taking the iron chelation rate as an observation index.
As can be seen from fig. 4, iron chelation rate increased first and then decreased as the temperature increased. At 4-30 ℃, the iron chelation rate is obviously increased along with the temperature rise; beyond 40 ℃, the iron sequestration rate decreases significantly as the temperature continues to rise. This is because the reaction does not proceed completely due to the excessively low temperature; the excessive temperature, although accelerating the reaction rate, greatly increases the energy consumption and is not favorable for the formation of the complex, and the increased temperature may cause the oxidation of the BSM, the decomposition of the complex, or other reactions, thereby decreasing the iron chelation rate. Therefore, the optimum temperature for the chelation reaction is 30 ℃.
D study of the influence of pH on the iron chelation Rate
Mixing BSM solution with FeCl3·6H2Mixing O solutions according to a volume ratio of 6:4, adjusting pH values to be 2, 3, 4, 5, 6, 7 and 8 under the conditions of reaction temperature of 30 ℃, rotation speed of 200rpm and reaction time of 5h, preparing seven groups of ferrous iron, and observing the influence of different pH values on the BM-Fe iron chelation rate by taking the iron chelation rate as an observation index.
FIG. 5 shows that at pH 2, the iron chelation rate is only 5%, probably because a large number of hydronium ions in the solution compete with metal ions for reactive sites, and also because the solution is strongly acidic, phenolic hydroxyl groups, carboxyl groups, amine groups, etc. in the BSM molecule are positively charged due to protonation, and are Fe3+Generate strong electrostatic repulsion between the two to lead Fe3+Difficulty in approaching BSM, resulting in Fe3+The chelation rate is almost zero; the chelating rate is increased along with the increase of the pH value, because the acidity of the solution is weakened, and the active sites in the adsorbent, which are complexed with hydronium ions, are gradually exposed to be beneficial to Fe3+Chelating; the pH value is more than 4 and then the whole body is in a descending trend, probably because the upper limit of iron ion micro-precipitation is exceeded, metal ions can form oxide precipitation; after pH is greater than 8, OH-in solution competes with BSM for Fe3+Free Fe in solution3+The concentration becomes smaller, resulting in a decrease in the chelation rate, and therefore, the investigation is not continued. Thus acidity chelates Fe to BSM3+With a major impact, the optimum pH was 3.
E study of the influence of rotational speed on iron chelation Rate
Mixing BSM solution with FeCl3·6H2Mixing O solutions according to a volume ratio of 6:4, controlling the rotating speed to be 0, 100, 200, 300 and 400rpm under the conditions that the pH value is 5, the reaction temperature is 30 ℃ and the reaction time is 5 hours, preparing five groups of melanin iron, and observing the influence of different rotating speeds on the BM-Fe iron chelation rate by taking the iron chelation rate as an observation index.
As can be seen from fig. 6, whether stirring has a significant effect on the iron chelation rate or not, the iron chelation rate was significantly increased after magnetic stirring, but the maximum iron chelation rate was observed within the test range at a rotation speed of 300 rpm. Therefore, the optimum rotation speed is selected to be 300 rpm.
Example 2
On the basis of a single-factor experiment, a response surface method is used for optimizing the preparation process. The chelating ratio (%) of BM-Fe wasAnd (4) carrying out Box-Behnken Design and response surface analysis experiments on the response values by using Design Expert 10 statistical software. Selecting the volume ratio X1Reaction time X2(h) Reaction temperature X3(℃)、PH X4Four factors, three levels were designed for the experiments, as shown in table 2.
Table 2 experimental design table of response surface
According to the response surface experiment result, a Design Expert 10 software is utilized to carry out multiple quadratic regression fitting on the relationship between the black sesame melanin-nano iron chelation rate Y and the corresponding volume ratio X1, reaction time X2(h), reaction temperature X3 (DEG C) and pH X4, and a model of a polynomial regression equation is obtained, 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
the results of the analysis of variance of each factor can be seen from table 5, when the chelation rate of BM-Fe is taken 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 significant and has statistical significance; meanwhile, the value P 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, no mismatching factor exists, the experimental error is small, and the model is successfully established. According to the P value, the main and secondary sequences of the factors influencing the BM-Fe chelating rate are as follows: volume ratio (X)1)＞pH(X4) Reaction temperature (X)3) Reaction time (X)2) (ii) a Interaction item X1X2、X1X3、X1X4、X2X3、X2X4、X3X4All P values of (A) are greater than 0.05, indicating interactionThe effect of the item was not significant, indicating that the four factors were not interacting. Through software analysis, the optimal theoretical preparation process parameters of BM-Fe are as follows: the volume ratio is 3.988: 4, the reaction time is 4 hours, the reaction temperature is 50 ℃, and the pH value is 3; considering the convenience and feasibility of actual 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 value is 3.
TABLE 5 regression equation analysis of variance results
Experimental example 1
This experimental example was conducted on the BM-Fe and BSM prepared under the optimum conditions obtained in example 1-2 by subjecting BSM and BM-Fe to infrared spectroscopy experiments, respectively, by grinding them with potassium bromide (KBr) in an agate pot, preparing wafers, respectively, measuring by a Fourier transform infrared spectrometer (FT-IR) to obtain infrared spectrograms, and comparing them.
As a result, as shown in FIG. 7, the absorption peak of BSM was mainly at 3365cm-1、2930cm-1、1625cm-1、1425cm-1Several groups of absorption peaks; 3365cm-1The absorption peak is very broad and strong, which indicates that BSM contains hydroxyl (-OH) and amino (-NH2), and the region also belongs to the stretching vibration of carbonyl (-C ═ O-), 2930cm-1The small and sharp absorption peak is caused by C-H stretching vibration in alkane structure, 1625cm-1、1425cm-1The strong absorption peak is caused by the vibration of the aromatic ring, and is a vibration expansion absorption peak of the aromatic ring C ═ C; 1625cm-1The strong absorption peak indicates the existence of carbonyl in the molecule, and 3400cm-1The strong absorption peaks at (A) together indicate the presence of a-COOH structure, 810-525cm-1The absorption peak is weaker, which shows that the aromatic ring is substituted, the aromatic hydrogen content is less, and a conjugated system is formed. 34 in BM-Fe compared to BSM00-3000cm-1、1500cm-1The infrared absorption peaks of the left, right and fingerprint regions confirm the existence of BSM, and partial absorption peaks are weakened, which indicates that carboxyl, hydroxyl and amino participate in the chelation of metal ions. At 3365cm-1The absorption peak at the wavelength is weakened, which shows that the strength of-OH or amino is reduced, the N atom of the amino can be subjected to coordination reaction or even disappear, and the stretching vibration frequency of the-N-H group is reduced, which shows that the iron ions are chemically combined with the BSM, the change of a plurality of characteristic peaks can reflect that the BSM and ferric trichloride hexahydrate have no great influence on the whole structure of the BSM after reaction, but the change of the peak values of a plurality of characteristic absorption peaks can show that Fe is contained in the BSM3+Mainly has complexation with amino, carboxyl and phenolic hydroxyl in BSM.
Experimental example 2
In this example, the surfaces of BM-Fe and BSM prepared under the optimal conditions obtained in example 1-2 were observed, and before the experiment, BSM and BM-Fe were placed on a sample stage using a double-sided conductive adhesive, subjected to a gold-spraying treatment, and then observed under a scanning electron microscope. FIG. 8 is an SEM image of BSM and BM-Fe, where A, C is BSM and B and D are BM-Fe.
It can be seen from fig. 8 that the two microscopic morphologies are significantly different, the BSM has a cubic block structure, the structure is obvious, no attachment is on the surface, and the morphology is more regular. BM-Fe is more aggregated, is flaky, is mostly irregular in shape and has a rough surface, and the result is probably that the surface appearance of BM-Fe is changed due to the change of some groups and the change of the internal structure after the reaction of BSM and iron.
Experimental example 3
In this experimental example, BM-Fe and BSM prepared under the optimum conditions obtained in example 1-2 were subjected to dynamic light scattering particle size analysis, and the BSM and BM-Fe prepared were diluted by the same factor and the particle size was measured using a dynamic light scattering instrument. FIG. 9 is a particle size diagram of BSM and BM-Fe of the present invention, wherein A is BSM and B is BM-Fe.
As shown in FIG. 9, the average particle size data of the two samples are 844.9nm and 294.3nm, respectively. The particle size of BM-Fe is significantly smaller than that of BSM, probably because the iron ions are combined with carboxyl and hydroxyl on BSM to generate structural folding, so as to form nano-carriers of the iron ions, and the particle size of BM-Fe is reduced.
Experimental example 4
This example was conducted to test the antioxidant property of BM-Fe prepared under the optimum conditions obtained in example 1-2.
(1) ABTS free radical scavenging assay
ABTS ionic liquid is prepared by mixing 7mM ABTS and 2.45mM potassium persulfate solution in dark place for 12-16h, and before use, the absorbance value of the ABTS ionic liquid at 734nm is adjusted to 0.7 +/-0.02 by using ethanol. In the reaction, each reagent is tested according to a sample adding sequence table, which is detailed in table 3, wherein BSM and BM-Fe solution are respectively diluted in half to 5 concentrations (1.25, 2.5, 5, 10, 20 μ g/mL) in turn, and are subjected to vortex oscillation reaction for 6min in a dark place at room temperature, and the absorbance value is measured at 734 nm. The positive control is the same concentration of Vc, and the scavenging capacity of ABTS free radicals is calculated according to the following formula:
in the formula: a. the1The absorbance value of the sample after reacting with ABTS free radicals; a. the2The absorbance value of the control group of absolute ethyl alcohol after reaction with ABTS free radicals; a. the0Initial absorbance values for ABTS free radical solutions.
TABLE 3 sample addition sequence chart
As is clear from FIG. 10, the inhibition rates of BSM, BM-Fe and Vc on ABTS all showed a certain concentration dependence. The removal efficiency of BSM and BM-Fe is obviously higher than that of Vc group. Exhibits excellent effects at very low concentrations, and is excellent in oxidation resistance, and the BSM molecule has oxidizing (o-quinone) and reducing (o-hydroquinone) groups and can remove active oxygen or active nitrogen radicals by losing electrons or capturing electrons during electron transfer by interaction with radicals. In addition, the functional groups with different oxidation resistance can be reduced mutually to generate a synergistic effect, so that the oxidation resistance of the BSM is stronger. And BM-Fe and BSM have equivalent clearing effect, which shows that the BSM and ferric trichloride react to better retain the antioxidant activity of the BSM.
(2) DPPH radical scavenging test
Accurately weighing 25.6mg of DPPH free radical, using absolute ethyl alcohol to fix the DPPH free radical in a 100mL volumetric flask, shaking up and storing the DPPH free radical in a dark place for later use. In the reaction, each reagent is tested according to a sample adding sequence table, wherein BSM and BM-Fe solutions are respectively diluted in half to 5 concentrations (12.5, 25, 50, 100 and 200 mu g/mL) in turn, VC solution with the same concentration is used as positive control, the reaction is carried out for 30min in a shading way at room temperature, and the absorbance value A is measured at the wavelength of 517 nm. The DPPH radical clearance is calculated as follows:
TABLE 4 sample addition sequence chart
As can be seen from FIG. 11, the inhibition rates of both BSM and BM-Fe on DPPH showed a certain concentration dependence. However, the removal efficiency of BSM and BM-Fe is significantly lower than that of the Vc group. When the concentration is 200 mug/mL, the clearance rate can reach about 40 percent, and the antioxidation effect is good. BSM and its derivatives may be directly trapped or bound to DPPH to scavenge DPPH radicals, several derivatives of BSM have lower clearance than BSM, probably because the amino acids and BSM, when acting, convert the diphenol/benzoquinone structure to lose electrons, reducing their radical scavenging activity. And BM-Fe scavenging efficiency comparable to BSM.
Generally, due to iron deficiency, the activity of iron-containing enzymes playing an important role in regulating various cellular processes in vivo is reduced, and the reduction of the activity of the enzymes seriously affects the normal metabolism of the body, leads to the increase of the oxidation level and increases the oxidative stress, thereby causing 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 well continues the strong antioxidation of BSM, and BM-Fe as a potential novel iron supplement agent has positive treatment effect on related diseases caused by anemia.
Experimental example 5
This example was conducted to perform an in vitro iron elution test on BM-Fe prepared under the optimum conditions obtained in example 1-2.
(1) Simulated gastric juice
20mg of BM-Fe was accurately weighed and dissolved in 100mL of hydrochloric acid solution (0.1moL/L), reacted at 37 ℃ under magnetic stirring at 100rpm for 2 hours, samples were taken every 0.5 hour for 3mL at a time, and the same volume of hydrochloric acid solution was added. After the reaction is finished, filtering the sample solution by a 0.45 mu m microporous membrane, respectively taking 2mL of each filtrate in a 50mL volumetric flask after the filtration, and then measuring the absorbance value at 510nm after the operation steps such as iron content measurement are finished.
As can be seen from FIG. 12, the BM-Fe is released at about 45% after 30min in gastric juice, which is probably due to the strong acid condition of gastric juice increasing the content of free iron ions released by BM-Fe. And when the time is 2 hours, the iron release rate of BM-Fe exceeds 80%.
(2) Simulated intestinal fluid
Accurately weighing 20mg BM-Fe, dissolving in 100mL buffer solution with pH 6.8, magnetically stirring at 37 deg.C and 100rpm for 4h, sampling every 0.5h, and adding 3mL buffer solution at the same time. After the reaction is finished, filtering the sample solution by a 0.45 mu m microporous membrane, respectively taking 2mL of each filtrate in a 50mL volumetric flask after the filtration, and then measuring the absorbance value at 510nm after the operation steps such as iron content measurement are finished.
As can be seen from FIG. 12, BM-Fe also has a high release rate in the intestinal fluid environment, and at 2h, the release rate of BM-Fe reaches 70%, and since iron ions are mainly absorbed in the upper part of duodenum and jejunum, the iron in BM-Fe has a good bioavailability. All experiments in this case were repeated 3 times and the results are expressed as mean X ± SD. The graph was plotted using Origin 2018 and the significance analysis was performed using SPSS 24.0, where different lower case letters indicate significant differences (P < 0.05).
As can be seen from the above experimental examples 1-5, iron successfully reacted with BSM; the dissolution rate of the iron element in the simulated gastrointestinal environment of BM-Fe is higher, which shows that the BM-Fe has good bioavailability in the gastrointestinal environment and better keeps the excellent characteristics of oxidation resistance and the like of melanin.
Wherein, the infrared result shows that carboxyl, hydroxyl and amino groups of the BSM participate in the chelation of iron ions; an antioxidation experiment shows that BM-Fe well continues the excellent characteristic of BSM antioxidation; the in vitro iron dissolution experiment result shows that BM-Fe is favorable for body absorption.
The existing iron-supplementing chelate compounds have been proved to have good blood-supplementing effect, but no attention is paid to the aspects of oxidation resistance and the like. Therefore, BM-Fe complex is expected to be developed into a novel multifunctional iron supplement with good activity, and has great prospect in biology, medicine and even application of novel functional food which has potential positive influence on health besides basic nutrition.
The above description is only a preferred embodiment of the present invention, and is not intended to limit the design of the present invention, and all equivalent changes made in the design key point of the present invention fall within the protection scope of the present invention.
Claims (8)
1. A preparation method of melanin iron with antioxidant activity is characterized in that a iron trichloride method is adopted to prepare the melanin iron, and the specific steps are as follows:
preparing black sesame melanin into BSM solution with a certain concentration, and adding FeCl according to a certain proportion3·6H2Mixing O solutions, adjusting pH, optimizing chelating conditions by a response surface method, reacting under the conditions of constant temperature and constant speed water bath stirring in a dark and normal pressure environment, centrifuging for 5min after the chelating reaction is finished, taking a precipitate, washing twice, and performing vacuum freeze drying to obtain the ferrous black.
2. The method for preparing melanin iron with antioxidant activity as claimed in claim 1, wherein the method for preparing melanin iron adopts response surface method to optimize the chelating condition of melanin iron, takes iron chelating rate as an investigation index, and takes BSM solution and FeCl as research indexes3·6H2The volume ratio, the reaction time, the reaction temperature, the pH value and the rotating speed of the O solution are considered factors, a single-factor experiment and a response surface experiment are designed in sequence, and the optimal preparation conditions are analyzed.
3. The method of claim 2, wherein the method comprises mixing BSM solution with FeCl3·6H2The volume ratio of the O solution is 4:4, 5:4, 6:4, 7:4 and 8: 4.
4. The method for preparing melanin iron having antioxidant activity according to claim 2, wherein the reaction time is 1-6h, the reaction temperature is 4-60 ℃, the pH is 2-8, and the rotation speed is 0-400 rpm.
5. The method of claim 1, wherein the concentration of the BSM solution is 0.4 mg/mL.
6. The method of claim 1, wherein the FeCl is FeCl3·6H2The concentration of the O solution was 0.6 mg/mL.
7. The method according to claim 1, wherein the mixture is centrifuged at 8000rpm for 5 min.
8. A melanin iron having an antioxidant activity, which is prepared by the method for preparing a melanin iron having an antioxidant activity according to any one of claims 1 to 8.
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| CN202111179686.1A CN113875990B (en) | 2021-10-11 | Ferrous melanin with antioxidant activity and preparation method thereof |
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