Corn polysaccharide zinc complex and its preparation method and application

Abstract

The invention discloses a corn polysaccharide zinc complex and a preparation method and application thereof, and belongs to the field of functional food and biomedicine technology. The preparation method specifically comprises the following steps: corn kernels are pretreated to obtain defatted corn flour, then water extraction and alcohol precipitation, drying and extracting corn polysaccharides, deproteinizing the corn polysaccharides by the Sevag method, and using the deproteinized corn polysaccharides and zinc chloride as raw materials to prepare corn polysaccharide zinc complexes. The invention can simultaneously have the physiological functions of corn polysaccharides and zinc, not only has a synergistic effect in regulating immunity, but also can effectively improve bioavailability, making it a zinc supplement with less side effects and capable of improving body immunity, suitable for people of all ages, simple production process, solves the problems of low absorption efficiency and single product of existing zinc supplements, and also has the advantages of low price and easy availability of raw materials compared with other polysaccharide zinc complexes.

Description

Technical Field

[0001] The invention relates to the technical field of functional food and biomedicine, and more specifically to a corn polysaccharide zinc complex and a preparation method and application thereof. Background Art

[0002] Corn is a widely grown crop worldwide. Due to its high yield, easy availability and low price, more and more researchers have studied the components and pharmacological effects of corn.

[0003] As one of the main active ingredients in corn, polysaccharides have multiple activities such as anti-oxidation, enhancing immunity, protecting the liver, regulating gastrointestinal function, etc. Most of their structures contain active groups such as hydroxyl, amino and negative oxygen groups, which are easy to form complexes with various metal ions through chemical and physical adsorption, such as zinc, copper, selenium, iron and calcium. These metal ions are all trace elements necessary for the human body. The combined complexes will exert better biological effects. At present, researchers have extracted natural polysaccharide trace element complexes from plants, but the trace element content in these complexes is very low, and the plant growth time is relatively long. Therefore, high-efficiency, high-trace element content artificial synthetic polysaccharide trace element complexes have attracted widespread attention.

[0004] Zinc is mainly distributed in bones, muscles and plasma. It can promote the normal development of organs such as skin and bones, maintain digestion and metabolism, and plays an important role in the process of immune regulation. It is known as the "element of life". However, the human body cannot synthesize zinc by itself and can only obtain it through diet. If a developing child is zinc deficient, it will show poor appetite, slow growth and development, and will also induce anorexia and dwarfism; zinc deficiency in adults will lead to decreased immunity and be more susceptible to influenza virus infection than healthy people; zinc deficiency in the elderly will cause fatigue, dizziness, nausea and weight loss. Due to the important role of zinc, various zinc supplements have emerged. Although the existing zinc supplement preparation technology is relatively mature, the product still has some defects, such as low bioavailability, long-term use will irritate the gastrointestinal tract, and even some products with high zinc content will antagonize the absorption of nutritional factors such as iron and calcium, which are not suitable for young children and the elderly. With the development of science and technology, biological zinc has gradually been accepted by consumers. The absorption rate and activity of this type of product are relatively good, but there is a shortcoming that the product is relatively single.

[0005] Therefore, providing a zinc supplement with high bioavailability, wide applicable range, low toxicity and side effects, and significant immune-enhancing effect is an urgent problem to be solved by those skilled in the art. Summary of the invention

[0006] In view of this, the present invention provides a preparation method and application of a corn polysaccharide zinc complex.

[0007] In order to achieve the above object, the present invention adopts the following technical solution:

[0008] A method for preparing a corn polysaccharide zinc complex comprises the following steps:

[0009] (1) corn kernels are pretreated to obtain defatted corn flour;

[0010] (2) extracting defatted corn flour with water, precipitating with alcohol and drying to obtain crude corn polysaccharides;

[0011] (3) deproteinizing the crude corn polysaccharide to obtain corn polysaccharide;

[0012] (4) preparing a corn polysaccharide solution, adding zinc chloride solution thereto and mixing thoroughly, adjusting the solution to alkalinity with NaOH solution, stirring and reacting at 50-60° C., and after the reaction is completed, precipitating with alcohol, centrifuging and re-dissolving the precipitate, dialyzing and collecting the dialysate, and freeze-drying to obtain a corn polysaccharide zinc complex.

[0013] Since polysaccharides and metal ions can react chemically under certain specific conditions, and the synthesized new compounds can simultaneously exert the biological activities of the polysaccharide and the metal element, polysaccharide metal complexes have been widely used in the fields of chemical industry, medicine, agriculture, etc. The structures, components, and molar ratios of different polysaccharides are different. It is precisely because of the diversity of these differences that even if different polysaccharides react with the same metal ions, the reaction conditions are not exactly the same. According to the coordinated electron theory, the corn polysaccharide zinc complex of the present invention is a coordination reaction between the lone pair electron groups provided by the hydroxyl groups in the polysaccharide and the zinc element, and the COOH on the uronic acid component contained therein can release H through a replacement reaction. + , and then form a polysaccharide zinc complex, and the pH is one of the main factors affecting the zinc content in the complex, which can determine the surface charge of the polysaccharide and the form of metal ions. Under alkaline conditions, the OH in the solution - As the concentration increases, it neutralizes the H + , which increases the number of negatively charged active sites on the polysaccharide surface, and is beneficial to complexation. However, when the solution is a strong alkaline environment, metal ions may form precipitation, thereby preventing the formation of complexes. Therefore, in order not to introduce other impurities, the present invention uses NaOH solution to strictly control the pH value.

[0014] Preferably: the specific operation of pre-treatment of corn kernels in step (1) is:

[0015] (11) crushing fresh corn kernels, and passing the resulting powder through a 24-50 mesh sieve to obtain corn meal;

[0016] (12) Corn meal is defatted by reflux with 5 times anhydrous ethanol for 2-3 times, each time for 2-3 hours, and then filtered and dried at 20-30°C to obtain defatted corn meal.

[0017] Preferably: the specific operation of extracting corn crude polysaccharides in step (2) is:

[0018] (21) Soak defatted corn flour in 40-45°C distilled water at a liquid-to-solid ratio of 20-24:1 for 30-40 min, set the heating temperature to 88-95°C, and extract for 2-3 h. After the extraction is completed, filter at 86-90°C, repeat the extraction twice, combine the filtrate, centrifuge at 20-25°C for 10-15 min, collect the supernatant and concentrate under reduced pressure to obtain corn polysaccharide concentrate;

[0019] (22) Anhydrous ethanol is added to the corn polysaccharide concentrate to make the ethanol volume concentration reach 80-85%, and the precipitate is collected by centrifugation after standing for 24-48 hours, and then vacuum dried to obtain crude corn polysaccharide.

[0020] Preferably: the specific operation of removing protein from corn crude polysaccharides in step (3) is:

[0021] A corn crude polysaccharide solution with a mass concentration of 10-20% is prepared, and 1 / 4-1 / 5 volume of Sevag reagent (chloroform: n-butanol = 4:1, V / V) is added, and the solution is fully shaken for 25-30 minutes. Under room temperature, the solution is centrifuged at a speed of 3000-3500 r / min for 15-20 minutes, and the supernatant is retained and dialyzed for 48-72 hours. The above operation is repeated 4-5 times, and the supernatant is concentrated and freeze-dried to obtain corn polysaccharide.

[0022] Preferably: the specific operation of preparing the corn polysaccharide zinc complex in step (4) is:

[0023] (41) preparing a corn polysaccharide solution with a mass concentration of 7-10 mg / mL, and slowly adding a 2-4 mg / mL zinc chloride solution with a pH of 3.0, the volume ratio of the corn polysaccharide solution to the zinc chloride solution is 1:2-1:3, stirring for 5-8 min, adjusting the pH to 8.0-10.0 with a 0.5 mol / L NaOH solution, and magnetically stirring at 50-60 °C for 48-72 h to obtain a pre-product;

[0024] (42) 4-5 times the volume of anhydrous ethanol was added, and the mixture was centrifuged at 5000 r / min for 10 min. The precipitate was redissolved and placed in a dialysis bag with a cutoff of 3500 Da for 48-72 h. The dialysate was collected and freeze-dried to obtain the corn polysaccharide zinc complex.

[0025] The invention also discloses the application of the corn polysaccharide zinc complex in the preparation of zinc supplementing medicines, immunity enhancing medicines or functional foods.

[0026] Furthermore, the zinc supplement and immune enhancement drug are in the form of tablets, oral liquids, capsules, granules or pills.

[0027] It can be seen from the above technical solution that, compared with the prior art, the beneficial effects achieved by the present invention are:

[0028] (1) It can simultaneously possess the physiological functions of corn polysaccharides and zinc.

[0029] (2) It can be used as a zinc supplement. Compared with other zinc supplement products, it not only has high bioavailability and less gastrointestinal irritation, but also can be taken for a long time. It can be taken by people of different ages, such as the elderly and children, and is suitable for a wider range of people.

[0030] (3) The corn polysaccharide and zinc in the present invention can work synergistically to significantly enhance the body's regulatory effect on immune function.

[0031] (4) The raw materials are cheap and easy to obtain, the product cost is low, and the product is stable and easy to preserve. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0033] Figure 1 The attached figure is a graph showing the results of the single factor test in Experiment 1;

[0034] Figure 2 The attached figure is a 3D surface plot of the response surface in Experiment 1;

[0035] Figure 3 The attached figure is a graph showing the results of the single factor test in Experiment 2;

[0036] Figure 4 The attached figure is an HPLC chart of the monosaccharide composition analysis of corn polysaccharide in Experiment 3; wherein, 1. Man; 2. Rha; 3. GlcA; 4. GalA; 5. Glc; 6. Gal; 7. Xyl; 8. Ara; 9. Fuc;

[0037] Figure 5 The attached figure is a UV-visible spectrum analysis diagram in Experiment 5;

[0038] Figure 6 The attached figure is the infrared spectrum of Experiment 6;

[0039] Figure 7 The accompanying figure is an X-ray powder diffraction pattern in Experiment 7;

[0040] Figure 8 The attached figure is a scanning electron microscope image from Experiment 8;

[0041] Fig. 9 The attached figure is the energy spectrum of corn polysaccharide in Experiment 9;

[0042] Fig.10 The attached figure is the energy spectrum of the corn polysaccharide zinc complex in Experiment 9;

[0043] Fig.11 The attached figure is a blood drug concentration-time curve in Experiment 10;

[0044] Fig.12 The attached figure is a graph showing the weight change trend of mice in Experiment 11. DETAILED DESCRIPTION

[0045] The technical solution of the present invention is further described in detail below through embodiments and in conjunction with the accompanying drawings.

[0046] Example 1

[0047] (1) fresh corn kernels are mechanically crushed and then passed through a 24-mesh sieve to obtain corn meal, 5 times the amount of anhydrous ethanol is added, and reflux defatting is performed twice, each time for 2 hours, and then filtered at 30° C. and dried to obtain defatted corn meal;

[0048] (2) adding 22 times the amount of distilled water to defatted corn flour, heating to 40° C. and soaking for 30 minutes, extracting at 90° C. for 2.5 hours, filtering at 88° C., repeating the above operation, combining the two filtrates, centrifuging at 20° C. and 3500 r / min for 10 minutes, collecting the supernatant and concentrating under reduced pressure to obtain corn polysaccharide concentrate; adding anhydrous ethanol to the polysaccharide concentrate to make the ethanol volume concentration reach 85%, standing for 48 hours, centrifuging and collecting the precipitate, and vacuum drying to obtain corn polysaccharide concentrate;

[0049] (3) Weigh corn crude polysaccharide and dissolve it in distilled water to prepare a crude polysaccharide solution with a mass concentration of 10%, add 1 / 4 volume of Sevag reagent (chloroform: n-butanol = 4:1, V / V), shake thoroughly for 30 minutes, centrifuge at room temperature and 3500 r / min for 15 minutes, retain the supernatant, dialyze for 72 hours, repeat the above operation 4 times, concentrate the supernatant, and freeze-dry to obtain corn polysaccharide;

[0050] (4) Weigh 90 mg of corn polysaccharide and dissolve it in 10 mL of distilled water, slowly add 3 mg / mL zinc chloride solution with pH = 3.0, the volume ratio is 1:2.5, stir for 5 min, adjust the pH to 9.0 with 0.5 mol / L NaOH solution, and continue stirring at 60 ° C for 48 h. After the reaction is completed, add 4 times the volume of anhydrous ethanol, centrifuge at 5000 r / min for 10 min, take the precipitate and redissolve it, place it in a dialysis bag with a cutoff of 3500 Da and dialyze it for 48 h, collect the dialysate, and freeze-dry it to obtain the corn polysaccharide zinc complex.

[0051] Example 2

[0052] The mass concentration of the zinc chloride solution in step (4) is 2.5 mg / mL, and the remaining operations are the same as in Example 1.

[0053] Example 3

[0054] In step (4), the volume ratio of the corn polysaccharide solution to the zinc chloride solution is 1:2, and the remaining operations are the same as in Example 1.

[0055] Example 4

[0056] In step (4), 100 mg of corn polysaccharide was weighed and dissolved in 10 mL of distilled water, and the remaining operations were the same as in Example 1.

[0057] Example 5

[0058] In step (4), the pH is adjusted to 10.0 with 0.5 mol / L NaOH solution, and the remaining operations are the same as in Example 1.

[0059] Example 6

[0060] In step (2), the volume ratio of defatted corn flour to distilled water is 1:20, and the remaining operations are the same as those in Example 1.

[0061] Example 7

[0062] In step (3), a corn crude polysaccharide solution with a mass concentration of 20% is prepared, and the remaining operations are the same as those in Example 1.

[0063] Comparative Example 1

[0064] The mass concentration of the zinc chloride solution in step (4) is 1 mg / mL, and the remaining operations are the same as in Example 1.

[0065] Comparative Example 2

[0066] The volume ratio of the corn polysaccharide solution to the zinc chloride solution in step (4) is 1:4, and the remaining operations are the same as those in Example 1.

[0067] Comparative Example 3

[0068] In step (4), the pH is adjusted to 7.0 with 0.5 mol / L NaOH solution, and the remaining operations are the same as in Example 1.

[0069] Experiment 1 Optimization of corn polysaccharide extraction process by combining single factor experiment with response surface methodology

[0070] (1) Single factor test

[0071] Taking the extraction time of 2h, heating temperature of 90°C and liquid-to-solid ratio of 20:1 in step (2) as the basic test conditions, the effects of different extraction times (1, 2, 3, 4, 5h), heating temperatures (60, 70, 80, 90, 100°C) and liquid-to-solid ratios (12:1, 16:1, 20:1, 24:1, 28:1) on the extraction rate of corn polysaccharides were investigated, and the remaining operations were the same as in Example 1. The results are shown in Figure 1 .

[0072] (2) Response surface experiment

[0073] On the basis of single factor experiment, the three factors of extraction time (A), heating temperature (B) and solid-liquid ratio (C) were used to design response surface method to analyze factors and levels and optimize the extraction process of corn polysaccharides. See Table 1. The results of response surface experiment are shown in Tables 2, 3, Figure 2 .

[0074] Table 1 Response surface analysis experimental factors and levels

[0075]

[0076] Table 2 Response surface analysis experimental plan and experimental results

[0077] Experiment No. A B C Extraction rate (%) 1 -1 -1 0 4.69 2 1 -1 0 5.82 3 -1 1 0 5.26 4 1 1 0 5.25 5 -1 0 -1 4.64 6 1 0 -1 6.23 7 -1 0 1 5.65 8 1 0 1 5.64 9 0 -1 -1 4.22 10 0 1 -1 4.81 11 0 -1 1 4.81 12 0 1 1 5.66 13 0 0 0 6.83 14 0 0 0 6.53 15 0 0 0 6.40 16 0 0 0 6.86 17 0 0 0 6.61

[0078] Table 3 Significance test of regression equation coefficients

[0079]

[0080] The corn polysaccharide extraction rate values ​​in Table 2 were used for regression analysis of the independent variables extraction time, heating temperature and liquid-to-solid ratio, and the resulting regression simulation equation was as follows: Y = 6.65 + 0.3369A + 0.1785B + 0.2334C - 0.2844AB - 0.3984AC + 0.0654BC - 0.3647A 2 -1.03B 2 -0.7397C 2 As shown in Table 3, the model is meaningful and has a good fit.

[0081] Depend on Figure 2It can be seen that the optimal process conditions for extracting corn polysaccharides are: extraction time 2.6h, heating temperature 91.81℃, liquid-to-solid ratio 22.31:1: the polysaccharide yield under this condition is 6.44%. Combined with actual operation, the optimal extraction process is optimized to extraction time 2.5h, heating temperature 90℃, and solid-to-liquid ratio of 22:1.

[0082] Experiment 2 Single factor experiment and orthogonal experiment L9(3 4 ) Combined with optimization of the preparation process of corn polysaccharide zinc complex

[0083] (1) Single factor test

[0084] Taking the volume ratio of 1:2.5, pH=9, reaction temperature of 60°C and reaction time of 60h in step (4) as the basic experimental conditions, the effects of different volume ratios (1:1, 1:1.5, 1:2, 1:2.5, 1:3), pH values ​​(6.0, 7.0, 8.0, 9.0, 10.0), reaction temperatures (50, 55, 60, 65, 70°C) and reaction times (24, 36, 48, 60, 72h) on the zinc content in the corn polysaccharide zinc complex were investigated, and the remaining operations were the same as in Example 1. The results are shown in Figure 3 .

[0085] (2) Orthogonal test

[0086] Based on the single factor results, three levels of significant influence were selected for each factor, and L9 (3 4 ) Orthogonal test table, with the volume ratio of polysaccharide solution to zinc solution (A), reaction time (B), reaction temperature (C) and pH value of the solution during the reaction (D) as variables, taking the average value of zinc content for range analysis, optimizing the reaction conditions of corn polysaccharide zinc complex, the factors and levels of experimental design are shown in Table 4. The results of the orthogonal experiment are shown in Table 5.

[0087] Table 4 Orthogonal test factor level table

[0088]

[0089] Table 5 Orthogonal test L9(3 4 )Result table

[0090]

[0091]

[0092] Depend on Figure 3As shown in Table 5, the zinc content in the conditions described in the present invention is relatively high, that is, the optimal range, wherein the factors that have a greater impact on the zinc content are B>C>D>A, and the factor combination is: A2B2C3D1. The zinc content of the test group is the highest, up to 6.55%, that is, the optimal reaction conditions of the corn polysaccharide zinc complex are: the volume ratio of the polysaccharide solution to the zinc solution is 1:2.5, NaOH adjusts the solution pH to 9.0, and reacts at 60°C for 48h.

[0093] Experiment 3 Determination of monosaccharide components of corn polysaccharides

[0094] Chromatographic conditions: Instrument: Agilent 1260ⅡHPLC; Chromatographic column: Diamonsil C 18 (250mm×4.6mm, 5μm); column temperature: 35℃; mobile phase: 0.1mol / L phosphate buffer (pH=6.7)-acetonitrile (83:17, V / V); detection wavelength: 250nm; flow rate: 0.6mL / min; injection volume: 10μL; elution mode: isocratic elution.

[0095] Determination method: Weigh 9 kinds of monosaccharides to prepare a mixed monosaccharide standard solution with a concentration of 10 mg / mL for later use. Weigh 10 mg of the corn polysaccharide sample prepared in Example 1 into an ampoule, add trifluoroacetic acid to hydrolyze, add methanol to wash the hydrolyzate and spin dry, repeat the operation 3 to 4 times, add 200 μL of distilled water to dissolve, take 100 μL of sample solution and standard solution respectively, add 300 μL of 0.5 mol / L PMP-methanol solution and 150 μL of 0.3 mol / L sodium hydroxide solution in turn, vortex mix, react in a 70°C water bath for 60 minutes, cool to room temperature, add 0.3 mol / L hydrochloric acid solution to neutralize, extract 3 times with chloroform, take the supernatant and filter through a 0.22 μm filter membrane, and analyze by HPLC. Results are shown in Figure 4 (Figure A is a mixed monosaccharide standard, and Figure B is a corn polysaccharide sample).

[0096] Depend on Figure 4 It can be seen that the prepared corn polysaccharide is an acidic heteropolysaccharide composed of mannose, rhamnose, glucuronic acid, glucose, xylose and arabinose. The molar ratio of each monosaccharide can be calculated from the peak area to be 0.53:0.27:0.19:1:0.64:0.86.

[0097] Experiment 4 Determination of physical and chemical indicators

[0098] The corn polysaccharide zinc complexes prepared in Examples 1-7 and Comparative Examples 1-3 were taken and their Zn content was measured using a Phenom ProX spectrometer with an accelerating voltage set to 5 kV. The measurement results are shown in Table 6.

[0099] Table 6 Test results of physical and chemical indexes of corn polysaccharide zinc

[0100]

[0101]

[0102] From the results of Examples 1-4 and Comparative Examples 1-2, it can be seen that corn polysaccharide and Zn 2+ The degree of coordination is related to the mass concentration of ZnCl2 solution. The higher the ZnCl2 mass concentration, the higher the zinc content. However, when the concentration exceeds a certain range, the zinc content decreases. The reason may be that the reaction environment is weakly alkaline. When Zn 2+ If the concentration is too high, it will react with OH - Precipitation occurs, resulting in loss of zinc ions and affecting the coordination of the two.

[0103] From the results of Examples 1-5 and Comparative Example 3, it can be seen that pH value is the factor that affects the reaction between corn polysaccharide and Zn 2+ The important factor of coordination is that when the solution contains H + When there is too much Zn 2+ When binding to the hydroxyl or carboxyl groups in polysaccharides, + Produce competitive effect, hinder Zn 2+ Combined with polysaccharides, so in a weakly alkaline environment, polysaccharide molecules and Zn 2+ Easier to combine.

[0104] It can be seen from Examples 6-7 that the preparation conditions of corn polysaccharide can also affect the degree of coordination with zinc ions, but the impact is relatively small.

[0105] Experiment 5: UV-Vis Spectroscopy Analysis

[0106] Weigh 2 mg of each of the corn polysaccharide prepared in step (3) of Example 1 and the corn polysaccharide zinc complex prepared in step (4), dissolve them in 20 mL of distilled water to prepare a 0.1 mg / mL sample solution, use distilled water as a blank control, and perform UV-visible spectrophotometer scanning detection in the range of 200 to 800 nm. The test results are shown in Table 1. Figure 5 .

[0107] Depend on Figure 5 The results show that corn polysaccharide and corn polysaccharide zinc complex have no obvious absorption peaks at wavelengths of 260nm and 280nm, indicating that corn polysaccharide and its polysaccharide zinc complex do not contain nucleic acids and proteins, and no absorption peak of zinc chloride is found in corn polysaccharide zinc complex, indicating that corn polysaccharide and Zn 2+ After coordination, chemical bonds are formed, and this process is not a simple physical adsorption or aggregation.

[0108] Experiment 6 Infrared Spectroscopy Analysis

[0109] 2 mg of the corn polysaccharide prepared in step (3) and the corn polysaccharide zinc complex prepared in step (4) of Example 1 were weighed respectively, mixed with KBr in a ratio of 1:1, and pressed into tablets. The samples were analyzed by Alpha Centaurt FT / IR infrared spectrometer at 4000-400 cm -1 Spectrum scanning is performed within the range, and the test results are shown in Figure 6 .

[0110] Depend on Figure 6 As shown, corn polysaccharide is at 3421.10cm -1 The broad peak at 2912.69 cm is the stretching vibration absorption peak caused by OH. -1 The absorption peak of the asymmetric stretching vibration of CH in (-CH3) is 1629.55cm -1 The carboxyl group (-COO - ) is the stretching vibration absorption peak caused by C=O in the reaction. After the reaction with zinc, the OH vibration peak moves to 3403.74 cm -1 It is speculated that it may be due to Zn 2+ The coordination reaction with the -OH group occurred, and the CH vibration absorption peak shifted to 2906.19 cm -1 It is speculated that it is because Zn 2+ Replaces H in the functional group + The polysaccharide structure changes, causing its wavelength to shift to a shorter wavelength, and the C=O stretching vibration absorption peak shifts to 1621.84 cm -1 It is speculated that it is also due to Zn 2+ The coordination between corn polysaccharide and corn polysaccharide is 1016.30cm -1 The absorption peak at this position belongs to the vibration absorption peak of the pyran ring. After the corn polysaccharide zinc complex is formed, the absorption peak here is significantly enhanced. In summary, after the polysaccharide and zinc react, their basic skeleton structure is not destroyed. In this process, functional groups such as OH, CH, and C=O all participate in the reaction to generate new compounds, indicating that the preparation method of the present invention can successfully prepare corn polysaccharide zinc complexes.

[0111] Experiment 7 X-ray powder diffraction analysis

[0112] The corn polysaccharide prepared in step (3) and the corn polysaccharide zinc complex prepared in step (4) of Example 1 were spread on a sample plate, compacted with a glass slide, and placed in an X-ray powder diffractometer to analyze the amorphous properties of the samples at 5-50°. The test results are as follows: Figure 7 shown.

[0113] Depend on Figure 7It can be seen that corn polysaccharide has a weak diffuse broad peak at 15-30°, indicating that the internal crystallinity of the polysaccharide is very weak and exists in an unstable form, which is basically an amorphous structure; after complexing with zinc ions, two more obvious broad peaks appear at 13-27°, indicating that it has certain weak crystallinity inside. Compared with corn polysaccharide, the structure after complexing is more stable.

[0114] Experiment 8 Scanning electron microscopy analysis

[0115] The powders of the corn polysaccharide prepared in step (3) and the corn polysaccharide zinc complex prepared in step (4) of Example 1 were uniformly dispersed on a sample stage with double-sided conductive adhesive, and analyzed under a Phenom ProX scanning electron microscope with an accelerating voltage of 15 kV. The test results are shown in FIG. Figure 8 .

[0116] From the surface morphology, Figure 8 As shown in .A and 8.B, corn polysaccharide has an irregular flake structure with a large shape, an uneven surface, and no obvious holes; corn polysaccharide zinc complex has a dispersed fragment structure with a rough surface, clear grooves, and obvious holes. Figure 8 .C, 8.D. When the polysaccharide reacts with zinc ions, its surface becomes rough, indicating that zinc is bound to its surface. The method of the present invention can successfully prepare corn polysaccharide zinc complex.

[0117] Experiment 9 Energy Spectrum Analysis

[0118] The corn polysaccharide prepared in step (3) and the corn polysaccharide zinc complex prepared in step (4) of Example 1 were respectively taken, and the contents of C, O, and Zn in the samples were determined using a Perkin-Elmer 2400 analyzer. The test results of corn polysaccharide are as follows: Fig. 9 As shown, the test results of corn polysaccharide zinc complex are as follows Fig.10 shown.

[0119] Depend on Fig. 9 , Fig.10 The results of elemental analysis (%) are as follows:

[0120] Corn polysaccharide: C: 53.18%, O: 46.82%;

[0121] Corn polysaccharide zinc complex: C: 52.87%, O: 40.52%, Zn: 6.61%.

[0122] Experiment 10 Bioavailability Experiment

[0123] Sixteen SD rats weighing 180-220 g were selected, half male and half female, and randomly divided into 2 groups. The corn polysaccharide zinc and zinc gluconate prepared in Example 1 were given by intragastric administration at a single dose of 3.6 mg Zn / kg. The rats were fasted for 12 h before intragastric administration, and blood was collected from the ophthalmic venous plexus at 0, 1, 2, 3, 4, 5, 6, 8, 12, and 24 h after administration. The blood was centrifuged at 10000 r / min for 5 min, and 1 mL of the supernatant was drawn to detect the zinc content. The blood drug concentration-time curve was plotted, and the bioavailability was calculated.

[0124]

[0125] Where AUC represents the area under the plasma drug concentration-time curve, T and R represent the test reagent and reference reagent, respectively, and D represents the dosage.

[0126] The blood drug concentration-time curve of rats is shown in Fig.11 As shown. Fig.11 It can be seen that after oral administration of zinc gluconate and corn polysaccharide zinc complexes, they are rapidly absorbed into the blood. Zinc gluconate reaches a peak after 1 hour, and the zinc concentration in plasma drops to 1.33 mg / L at 3 hours, and then continues at this level. The corn polysaccharide zinc complex reaches the maximum concentration 3 hours after administration, drops to 1.76 mg / L at 6 hours, and then slowly decreases and gradually stabilizes. It is calculated that the zinc absorption of SD rats for corn polysaccharide zinc complex is higher than that of zinc gluconate, and the bioavailability is 80.17%. In addition, compared with zinc gluconate, the peak zinc concentration of corn polysaccharide zinc complex appears later, indicating that during the digestion process, corn polysaccharide zinc has a gentle effect on the release of zinc ions and is less irritating to the gastrointestinal tract than zinc gluconate.

[0127] Experiment 11 Experiment on the effect of corn polysaccharide zinc on immune function

[0128] Select 56 healthy Kunming mice, half male and half female. After 7 days of adaptive feeding, the mice were randomly divided into 7 groups: blank group, model group, zinc gluconate group (specification of zinc gluconate tablets: 70 mg / tablet, 10 mg / tablet in terms of Zn), corn polysaccharide group, zinc chloride group, corn polysaccharide + zinc chloride group, corn polysaccharide zinc complex group. Except for the blank group, the remaining groups were intraperitoneally injected with cyclophosphamide at a dose of 40 mg / (kg·d) for 3 consecutive days to induce an immunosuppressive model. According to the reference intake of nutrients for normal adults in China, the maximum daily intake of zinc for mice was calculated to be 5 mg / kg, corresponding to a zinc gluconate dosage of 35 mg / kg, a zinc chloride dosage of 10.4 mg / kg, and a corn polysaccharide zinc complex dosage (zinc content 6.58%) of 76 mg / kg. The corn polysaccharide and corn polysaccharide zinc complex prepared in Example 1 were selected for testing. From the 11th to the 21st day of the experiment, each group was given continuous oral administration according to the dosage shown in Table 7. The corn polysaccharide + zinc chloride group was given oral administration separately. After the polysaccharide was taken, the zinc chloride solution was continued to be given within 30 minutes.

[0129] Table 7 Grouping of mice and dosage

[0130]

[0131] Corn polysaccharide + zinc chloride group 71mg / kg + 10.4mg / kg

[0132]

[0133] 1. Effects on mouse body weight

[0134] Starting from the first day of administration, the body weight of the mice was recorded every 3 days.

[0135] 2. Effects on mouse immune organ index

[0136] After the experiment, the mice were killed by cervical dislocation, and their thymus and spleen were quickly removed and weighed, and the thymus index and spleen index of the mice were calculated.

[0137]

[0138] 3. Determination of immune factor content

[0139] The mouse blood was obtained and centrifuged at 3000 r / min for 10 min, and the supernatant was taken to obtain the serum. The concentrations of interleukin 6 (IL-6) and tumor necrosis factor (TNF-α) in the serum were determined strictly according to the instructions of the kit.

[0140] 4. Results

[0141] 4.1 Results of mouse weight changes

[0142] The weight of mice is positively correlated with their immunity. Fig.12 It can be seen that compared with the model group, the weight of mice in the corn polysaccharide and zinc chloride groups was restored, and the growth trend was the same as that of the zinc gluconate group (control drug), indicating that at this dose, supplementation of zinc or corn polysaccharide can restore the immunity of mice to a certain extent, which is an effective dosage. Comparing the corn polysaccharide + zinc chloride group with the corn polysaccharide zinc complex group, it can be seen that the weight growth rate and growth amount of mice in the complex group are significantly higher than those in the corn polysaccharide + zinc chloride group. It is speculated that this is because direct zinc ion supplementation is not easily absorbed, and the bioavailability of zinc is improved after the complex. Corn polysaccharides and zinc may regulate immunity through different pathways, producing a certain synergistic effect. Therefore, after the formation of the complex, it can significantly promote the growth of small weights of immunosuppressive by improving immunity.

[0143] 4.2 Results of mouse immune organ index determination

[0144] The thymus and spleen are important immune organs of the human body. The organ index can reflect the immune function of the body to a certain extent. As shown in Table 8, compared with the blank group mice, the thymus index and spleen index of the model group mice were significantly reduced, indicating that the autoimmunity of the mice was suppressed and the model was successfully established; compared with the model group, the immune organ index of the mice in the corn polysaccharide group and the zinc chloride group increased, indicating that at this dose, the supplementation of zinc and corn polysaccharides can have a certain protective effect on the thymus and spleen of acute immunosuppressed mice and restore their immune function, which is an effective dosage; comparing the mice in the corn polysaccharide + zinc chloride group and the polysaccharide zinc complex group, it can be seen that the change in the organ index of the mice in the complex group (the difference with the model group) is higher than the change caused by the administration of corn polysaccharide + zinc chloride, indicating that the combination does not physically superimpose the efficacy of the two, but can improve the absorption and bioavailability of zinc and corn polysaccharides in mice, so that the effect of improving immunity is better.

[0145] Table 8 Results of determination of immune organ index of mice in each group

[0146]

[0147] Note: *Compared with the model group (p﹤0.05); **Compared with the model group (p﹤0.01); ##Compared with the blank group (p﹤0.01)

[0148] 4.3 Results of immune response experiments

[0149] IL-6 and TNF-α are important inflammatory factors involved in immune regulation in the body. As shown in Table 9, compared with the blank group, the cytokine level of mice in the model group decreased, indicating that the body's immune function was disordered and the model was successfully established; compared with the model group, the serum IL-6 and TNF-α levels of mice in each administration group increased, indicating that at this dosage, supplementation of zinc (zinc gluconate, zinc chloride), corn polysaccharides and corn polysaccharide zinc complexes can promote the secretion of cytokines in immunosuppressive mice to varying degrees, thereby enhancing the nonspecific immune response of mice. Comparing the corn polysaccharide + zinc chloride group with the polysaccharide zinc complex group, it can be seen that the IL-6 and TNF-α levels of mice in the polysaccharide zinc complex group increased most significantly, and the increase was higher than the increase caused by the administration of corn polysaccharides and zinc chloride, which is consistent with the trend of the results under 4.2, indicating that the combination is not a simple physical superposition of the pharmacological effects of corn polysaccharides and zinc elements, but can work synergistically to effectively enhance the immunity of mice.

[0150] Table 9 Immune response experiment results

[0151]

[0152] Note: *Compared with the model group (p﹤0.05); **Compared with the model group (p﹤0.01); ##Compared with the blank group (p<0.01)

[0153] In summary, the present invention successfully prepared corn polysaccharide zinc complexes by combining corn polysaccharides with zinc ions, and analyzed its physicochemical properties by various characterization methods. The results show that compared with corn polysaccharides, corn polysaccharide zinc complexes have a more stable structure without destroying their original skeleton, and can simultaneously exert the biological activities of zinc ions and polysaccharides. From the perspective of regulating the body's immune function, corn polysaccharide zinc complexes are superior to zinc gluconate in terms of bioavailability and the ability to improve immunity; compared with the groups given corn polysaccharides or zinc chloride alone, the combination of the two can significantly enhance the immune function of mice, and the effect is better than the change caused by giving equal doses of corn polysaccharides and zinc elements separately, indicating that the two can produce a synergistic effect.

[0154] In this specification, each embodiment is described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the embodiments can be referred to each other. For the device disclosed in the embodiment, since it corresponds to the method disclosed in the embodiment, the description is relatively simple, and the relevant parts can be referred to the method part.

[0155] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. An application of a corn polysaccharide zinc complex in the preparation of an immune enhancing drug, characterized in that: The preparation method of the corn polysaccharide zinc complex comprises the following steps: (1) corn kernels are pretreated to obtain defatted corn flour; (2) extracting defatted corn flour with water, precipitating with alcohol and drying to obtain crude corn polysaccharides; (3) deproteinizing the crude corn polysaccharide to obtain corn polysaccharide; (4) preparing a corn polysaccharide solution, adding zinc chloride solution thereto and mixing thoroughly, adjusting the solution to alkalinity with NaOH solution, stirring and reacting at 50-60° C., and after the reaction is completed, precipitating with alcohol, centrifuging and re-dissolving the precipitate, dialyzing and collecting the dialysate, and freeze-drying to obtain a corn polysaccharide zinc complex.

2. The use of a corn polysaccharide zinc complex as claimed in claim 1 in the preparation of an immune enhancing drug, characterized in that: The corn kernel pretreatment in step (1) specifically comprises: (11) crushing fresh corn kernels, and passing the resulting powder through a 24-50 mesh sieve to obtain corn meal; (12) Corn meal is defatted by reflux with 5 times anhydrous ethanol for 2-3 times, each time for 2-3 hours, and then filtered and dried at 20-30°C to obtain defatted corn meal.

3. The use of a corn polysaccharide zinc complex as claimed in claim 1 in the preparation of an immune enhancing drug, characterized in that: The specific operation of step (2) is: (21) taking defatted corn flour and soaking it in 40-45° C. distilled water at a liquid-solid ratio of 20-24:1 for 30-40 min, setting the heating temperature to 88-95° C., and extracting it for 2-3 h; after the extraction is completed, filtering it at 86-90° C., repeating the extraction twice, combining the filtrate, centrifuging it at 20-25° C. for 10-15 min, collecting the supernatant and concentrating it under reduced pressure to obtain corn polysaccharide concentrate; (22) Anhydrous ethanol is added to the corn polysaccharide concentrate to make the ethanol volume concentration reach 80-85%, and the precipitate is collected by centrifugation after standing for 24-48 hours, and then vacuum dried to obtain crude corn polysaccharide.

4. The use of a corn polysaccharide zinc complex as claimed in claim 1 in the preparation of an immune enhancing drug, characterized in that: The specific operation of step (3) is as follows: prepare a corn polysaccharide solution with a mass concentration of 10-20%, add 1 / 4-1 / 5 volume of Sevag reagent, fully shake for 25-30 minutes, centrifuge at a speed of 3000-3500 r / min for 15-20 minutes at room temperature, retain the supernatant, dialyze for 48-72 hours, repeat the above operation 4-5 times, concentrate the supernatant, and freeze-dry to obtain corn polysaccharide.

5. Use of a corn polysaccharide zinc complex as claimed in any one of claims 1 to 4 in the preparation of an immune enhancing drug, characterized in that: The specific operation of step (4) is: (41) preparing a corn polysaccharide solution with a mass concentration of 7-10 mg / mL, and slowly adding a 2-4 mg / mL zinc chloride solution with a pH of 3.0, the volume ratio of the corn polysaccharide solution to the zinc chloride solution being 1:2-1:3, stirring for 5-8 min, adjusting the pH to 8.0-10.0 with a 0.5 mol / L NaOH solution, and magnetically stirring at 50-60 °C for 48-72 h to obtain a pre-product; (42) 4–5 volumes of anhydrous ethanol were added to the pre-product, and the mixture was centrifuged at 5000 r / min for 10 min. The precipitate was redissolved and placed in a dialysis bag with a cutoff of 3500 Da for 48–72 h. The dialysate was collected and freeze-dried to obtain the corn polysaccharide zinc complex.

6. The use of a corn polysaccharide zinc complex as claimed in claim 1 in the preparation of an immune enhancing drug, characterized in that: The immune enhancing drug is in the form of tablets, oral liquid, capsules, granules or pills.

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