Low-molecular pectin nano-selenium compound as well as preparation method and application thereof

Abstract

The invention discloses a low-molecular pectin nano-selenium compound and a preparation method and application thereof.The preparation method comprises the steps that sunflower pectin is subjected to enzymolysis to obtain pectin lysate, the lysate passes through 30 kDa and 10 kDa polyethersulfone membranes to obtain low-molecular pectin (SULMP) and pectin oligosaccharide (SUPO), potassium iodide, ascorbic acid and a tetravalent selenium source are added respectively, after a reaction is conducted, dialysis is conducted, and the low-molecular pectin nano-selenium compound is obtained. And freeze-drying the nano-selenium powder to obtain a powdery solid, so as to obtain the nano-selenium with two different molecular weights, namely, SULMP-SeNPs and SUPO-SeNPs. According to the preparation method, low-molecular pectin from sunflower heads is used as a stabilizer, the preparation condition of SULMP-SeNPs is optimized, and the particle size of the prepared low-molecular pectin nano-selenium compound is kept stable after the compound is stored at 4 DEG C for 8 weeks. The SULMP-SeNPs has good antibacterial and tumor inhibition effects, and the method is simple and convenient to operate and mild in reaction condition, and has the advantages of high reduction rate of high-valence selenium, controllable particle size of the obtained nano-selenium and the like.

Description

Technical Field

[0001] The invention belongs to the field of bioengineering, and specifically relates to a low-molecular pectin nano-selenium complex and a preparation method and application thereof. Background Art

[0002] Nanoselenium (SeNPs) is a single substance of selenium with nano-size characteristics. Because of its unique advantages such as high biological activity, low toxicity, high bioavailability, large specific surface area and high particle dispersibility, it has attracted more and more attention in food, agriculture, animal husbandry and aquaculture, and has played a series of roles in food and medicine related fields. However, SeNPs are very easy to aggregate and precipitate, turning into heat-stable gray single substance selenium, thus losing their biological activity. Polysaccharides, as a natural polymer, are composed of more than 10 monosaccharides connected in the form of glycosidic bonds. Natural or modified polysaccharides contain a variety of functional groups, such as hydroxyl, amino and carboxyl groups. These functional groups help to form intermolecular and intramolecular hydrogen bonds on the polymer chain, thereby stabilizing the prepared SeNPs by embedding. Recent studies have pointed out that the combination of Se and polysaccharides can effectively alleviate some of its own limitations, such as poor stability, large particles, low solubility and low bioavailability, and can enhance their biological activities, such as antibacterial and anti-tumor activities.

[0003] There are biological, physical and chemical methods for preparing polysaccharide nano-selenium. The biological method is further divided into biological cell secretion embedding method and direct embedding method of bioactive polysaccharides. Compared with other methods, direct embedding method of bioactive polysaccharides has the advantages of fast reaction speed, simple experimental operation and controllable product quality, so it has become the main method for preparing polysaccharide nano-selenium. At present, polysaccharides are widely used as embedding agents or dispersion media in the preparation of polysaccharide nano-selenium. The key to this method is to use ascorbic acid, glutathione and the like as reducing agents to convert sodium selenite or selenium dioxide into the zero-valent form of elemental selenium, and then interact with the active groups of polysaccharides to generate nano-selenium. In this process, by adjusting the reaction conditions, the controllable synthesis of nanoparticle morphology can be achieved. Summary of the invention

[0004] In view of the above-mentioned technical problems existing in the prior art, the purpose of the present invention is to provide a low-molecular-weight pectin nano-selenium complex and a preparation method and application thereof. The present invention utilizes pectin from sources such as hydrolyzed sunflower discs and obtains low-molecular-weight pectin through a polyethersulfone membrane to encapsulate and prepare nano-selenium. The preparation process is simple, fast and highly operable. The prepared nano-selenium has a small particle size and good stability. The particle size does not change significantly after being stored at 4°C for 8 weeks and no precipitation occurs. Compared with the pectin nano-selenium complex that has not been membrane-passed, the antibacterial and anti-tumor abilities of the low-molecular-weight pectin nano-selenium complex are greatly improved, and the problem that nano-selenium is unstable and easily spontaneously aggregates and precipitates, resulting in reduced biological activity and bioavailability is solved.

[0005] The technical solution adopted by the present invention is as follows:

[0006] A method for preparing a low molecular weight pectin nano-selenium complex comprises the following steps:

[0007] 1) Preparation of low molecular weight pectin lysate

[0008] The pectin is dissolved in ultrapure water at a material-liquid mass ratio of 1:50 to 1:200, the pH is adjusted to acidic, pectinase is added, and the temperature is maintained at 30 to 60°C for enzymatic hydrolysis. After the reaction is completed, the enzyme activity is immediately boiled to inactivate the enzyme, and the bottom precipitate is removed by centrifugation after cooling to room temperature, and the supernatant is collected as the enzymatic hydrolyzate; after filtering the enzymatic hydrolyzate, 0.5-2% dry yeast by volume of the filtrate is added to the filtrate, and the fermentation is carried out at 25-35°C for 25-45h, and the fermentation is carried out by centrifugation, and the supernatant is collected to obtain the fermentation liquid;

[0009] The fermentation broth was passed through a microfiltration device (30 kDa polyethersulfone membrane) to remove unhydrolyzed pectin, and then passed through a 10 kDa polyethersulfone membrane to remove small molecular weight polysaccharides to obtain a low molecular weight pectin (SULMP) filtrate, which was concentrated under reduced pressure using a rotary evaporator to obtain low molecular weight pectin SULMP, and the removed small molecular weight polysaccharides were recovered as pectin oligosaccharides (SUPO).

[0010] 2) Preparation of low molecular weight pectin nano-selenium complex

[0011] The low molecular weight pectin SULMP in step 1) is mixed with potassium iodide, ascorbic acid and a tetravalent selenium source, dissolved in water to prepare a solution, stirred and reacted in a water bath environment, and the obtained reaction solution is dialyzed to obtain a low molecular weight pectin nano-selenium complex solution, which is then freeze-dried into a powdery solid to obtain a low molecular weight pectin nano-selenium complex SULMP-SeNPs. SUPO-SeNPs and SP-SeNPs are prepared in the same way.

[0012] Furthermore, the pectinase is pectin lyase, and the enzyme activity concentration of the pectin lyase in the reaction solution of the enzymatic hydrolysis reaction is 5-15 U / mL, preferably 9-10 U / mL.

[0013] Furthermore, in step 1), the pH is adjusted to 4.0-6.0, preferably pH 5.5, the reaction temperature is 30-60° C., preferably 45° C., and the enzymatic hydrolysis time is 2-8 h, preferably 3 h.

[0014] Furthermore, in the mixed solution for water bath reaction in step 2), the concentration of low molecular weight pectin is 1.5-3.5 mg / mL, preferably 2.5 mg / mL, the concentration of ascorbic acid is 20-40 mM, preferably 30-35 mM, and the molar concentration of potassium iodide is 1-20 mM, preferably 10-15 mM.

[0015] Furthermore, the reaction temperature in step 2) is 25-35°C, preferably 30°C, the reaction time is 2-20h, preferably 8h, and the product needs to be left in the dark for 20-30h to age.

[0016] Furthermore, in step 2), the dialysis process includes transferring the reaction solution to a 2500-3500Da dialysis bag, controlling the dialysis temperature at 2-6°C in a light-proof environment, and continuing the dialysis for 2-4 days, preferably 3 days.

[0017] Furthermore, in step 2), the low molecular weight pectin nano-selenium complex solution is placed in a -20°C ± 2°C refrigerator for pre-freezing for 10-12 hours, and then the pre-frozen low molecular weight pectin nano-selenium complex is placed in a freeze dryer at -45 to -55°C for freeze drying for 30-40 hours, and the vacuum degree of the freeze dryer is above 0.09MPa.

[0018] The present invention also provides application of the low-molecular-weight pectin nano-selenium complex in the preparation of antibacterial or anti-tumor drugs.

[0019] Pectin lyase (PL) is a type of pectin depolymerase. Pectin lyase (PL) is an enzyme that cuts α-1,4-glycosidic bonds through β-trans elimination and degrades demethylated pectin to produce oligosaccharides of different molecular weights. Low molecular weight pectin is collected by microfiltration, and its rich unsaturated double bonds have a significant effect on the biological activity of oligosaccharide products.

[0020] Compared with the prior art, the beneficial effects of the present invention are mainly reflected in:

[0021] 1. SP and its enzymatic hydrolysis-derived SULMP and SUPO were used as embedding agents, and Vc was used as a reducing agent to convert Na 2 SeO 3Reduction formed uniform and stable SP-SeNPs, SULMP-SeNPs and SUPO-SeNPs, with an average particle size of 110.5nm for SULMP-SeNPs and 57.5nm for SUPO-SeNPs. When stored at 4°C, SULMP-SeNPs were more stable and remained clear and transparent without precipitation after 8 weeks of storage, effectively improving bioavailability.

[0022] 2. The SULMP-SeNPs obtained in the present invention have a strong inhibitory effect on both Gram-positive bacteria and Gram-negative bacteria, and also have a strong inhibitory effect on liver cancer HepG2 cells, and the inhibitory ability is positively correlated with the concentration.

[0023] 3. The present invention makes full use of agricultural waste resources of sunflower discs, better solves the problem of recycling sunflower discs, and reduces environmental pollution. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The effect of pectin concentration on the particle size of SULMP-SeNPs in Example 1;

[0025] Figure 2 This is a graph showing the effect of Vc concentration on the particle size of SULMP-SeNPs in Example 1;

[0026] Figure 3 This is a graph showing the effect of reaction temperature on the particle size of SULMP-SeNPs in Example 1;

[0027] Figure 4 This is a graph showing the effect of reaction time on the particle size of SULMP-SeNPs in Example 1;

[0028] Figure 5 This is a graph showing the effect of KI concentration on the particle size of SULMP-SeNPs in Example 1;

[0029] Figure 6 This is a graph of experimental results obtained by optimizing the optimal conditions for the minimum particle size of SULMP-SeNPs particle size concentration in Example 2.

[0030] Figure 7 This is a graph showing the particle size changes of the three nano-selenium complexes prepared in Example 3 when stored at 4°C.

[0031] Figure 8 is a scanning electron microscope image of SULMP-SeNPs in Example 4;

[0032] Fig. 9 is the FTIR image of SULMP-SeNPs in Example 4;

[0033] Fig.10is the XPS graph of SULMP-SeNPs in Example 4;

[0034] Fig.11 This is a graph showing the antibacterial effects of the three nano-selenium complexes on B. subtilis in Example 5;

[0035] Fig.12 This is a diagram showing the antibacterial effects of the three nano-selenium complexes on E. coli in Example 5;

[0036] Fig.13 This is a graph showing the antibacterial effects of the three nano-selenium complexes on S. aureus in Example 5;

[0037] Fig.14 This is a diagram showing the inhibitory effects of three nano-selenium complexes on HepG2 tumor cells in Example 6;

[0038] Fig.15 This is a graph showing the toxic effects of three nano-selenium complexes on L02 normal liver cells in Example 6;

[0039] Fig.16 The effect of three nano-selenium complexes on the ROS level changes of HepG2 tumor cells in Example 6

[0040] Fig.17 This is a graph showing the effects of the three nano-selenium complexes in Example 6 on the changes in ROS levels in L02 normal liver cells. DETAILED DESCRIPTION

[0041] The present invention will be further described below in conjunction with specific embodiments, but the protection scope of the present invention is not limited thereto.

[0042] The pectin in the embodiment of the present invention is sunflower pectin produced by Zhuhai Fanhai Biotechnology Co., Ltd.

[0043] The normal temperature in the present invention refers to 25-30°C.

[0044] The pectin lyase of the present invention is purchased from Cangzhou Xiasheng Enzyme Biotechnology Co., Ltd., and is in liquid form, with an enzyme activity of 600 U / mL, a temperature action range of 30-55° C., and a pH action range of 4.0-6.0.

[0045] Example 1: Preparation of low molecular weight pectin nano-selenium complex

[0046] According to the reaction conditions, a single factor experiment was carried out with the particle size of low molecular weight pectin nano-selenium complex as a reference indicator. The effects of five factors, including pectin concentration (1.5, 2, 2.5, 3 and 3.5 mg / mL), Vc concentration (20, 25, 30, 35 and 40 mM), reaction temperature (25, 30, 37, 50 and 60°C), reaction time (2, 4, 6, 8, 12 and 20 h), and KI concentration (1, 5, 10, 15 and 20 mM), on the particle size of low molecular weight pectin nano-selenium complex were studied.

[0047] The preparation method comprises the following steps:

[0048] 1) Pectin enzymatic hydrolysis method

[0049] Weigh 10g pectin (SP), add deionized water at a material-liquid mass ratio of 1:100 and mix well to obtain a pectin solution, add pectin lyase, the added volume of pectin lyase is 1.5% (v / v) of the total volume of the pectin solution, adjust to pH = 5.5, extract and hydrolyze in a constant temperature water bath, the reaction temperature is 45°C, and the reaction time is 3h. After the reaction is completed, boil and inactivate the enzyme immediately, cool to room temperature, centrifuge at 10000rpm for 15min at 4°C, and collect the supernatant to obtain the enzymatic solution. After filtering the enzymatic solution using a suction filter, take the supernatant and add 1% (supernatant volume) of dry yeast, ferment at 30°C for 36h, centrifuge at 10000rpm for 15min at 4°C, and collect the supernatant to obtain the fermentation liquid.

[0050] 2) Preparation of pectin lysate

[0051] The fermentation broth was passed through a microfiltration device (30 kDa polyethersulfone membrane) to remove the unhydrolyzed pectin, and then the small molecular weight polysaccharides were removed through a 10 kDa polyethersulfone membrane to obtain a low molecular weight pectin (SULMP) filtrate, which was concentrated under reduced pressure using a rotary evaporator to obtain low molecular weight pectin SULMP. At the same time, the removed small molecular weight polysaccharides were recovered to obtain pectin oligosaccharides (SUPO), and the raw material pectin (SP) was used as a control.

[0052] 3) Preparation of SP-SeNPs / SULMP-SeNPs / SUPO-SeNPs

[0053] The three kinds of pectins in step 2) are mixed with potassium iodide KI, ascorbic acid Vc and sodium selenite respectively, dissolved in water to form a solution, stirred and reacted in a water bath environment, and the obtained reaction solution was dialyzed (the reaction solution was transferred to a 3000Da dialysis bag, the dialysis temperature was controlled at 4°C and in a light-proof environment, and the dialysis was continued for 45 hours to obtain a low-molecular-weight pectin nano-selenium complex solution, and the low-molecular-weight pectin nano-selenium complex solution was placed in a -20°C refrigerator for pre-freezing for 10-12 hours, and then the pre-frozen low-molecular-weight pectin nano-selenium complex was freeze-dried in a freeze dryer at -50°C for 36 hours, and the vacuum degree was above 0.09MPa, and three kinds of pectin nano-selenium complexes were obtained.

[0054] According to the above-mentioned preparation method of low molecular weight pectin nano-selenium complex, taking the preparation of SULMP-SeNPs as an example, the following single factor experiment was conducted on the reaction conditions of SULMP-SeNPs in step 3):

[0055] 1. Effect of pectin concentration on the particle size of SULMP-SeNPs

[0056] In step 3), when the concentration of Vc in the reaction solution was 30 mM, the concentration of KI was 10 mM, the reaction temperature was 25°C, and the reaction time was 4 h, the particle sizes of SULMP-SeNPs were measured when the concentrations of pectin SULMP were 1.5, 2, 2.5, 3, and 3.5 mg / mL, respectively. Figure 1 As shown, when the SULMP concentration was 2.5 mg / mL, the particle size of SULMP-SeNPs was smaller.

[0057] 2. Effect of ascorbic acid solution concentration on the particle size of SULMP-SeNPs

[0058] In step 3), when the pectin concentration in the reaction solution was 2.5 mg / mL, the KI concentration was 10 mM, the reaction temperature was 25° C., and the reaction time was 4 h, the particle sizes of SULMP-SeNPs were measured when the Vc concentrations were 20, 25, 30, 35, and 40 mM, respectively. Figure 2 It is shown that when the concentration of ascorbic acid solution is 30-35 mM, the particle size of the nano-selenium complex remains at a small level.

[0059] 3. Effect of reaction temperature on the particle size of SULMP-SeNPs

[0060] In step 3), when the concentration of Vc in the reaction solution was 30 mM, the concentration of KI was 10 mM, the concentration of pectin was 2.5 mg / mL, and the reaction time was 4 h, the particle size of SULMP-SeNPs was measured at the reaction temperatures of 25, 30, 37, 50, and 60 °C, respectively. Figure 4 As shown, when the reaction temperature is 25-50°C, the particle size of SULMP-SeNPs remains at a small level.

[0061] 4. Effect of reaction time on the particle size of SULMP-SeNPs

[0062] In step 3), when the concentration of Vc in the reaction solution was 30 mM, the concentration of KI was 10 mM, the reaction temperature was 25°C, and the concentration of pectin was 2.5 mg / mL, the particle size of SULMP-SeNPs was measured when the reaction time was 2, 4, 6, 8, 12, and 20 h, respectively. Figure 3 As shown, when the reaction time is 8h, the particle size of the nano-selenium complex is the smallest.

[0063] 5. Effect of KI concentration on the particle size of SULMP-SeNPs

[0064] In step 3), when the concentration of Vc in the reaction solution was 30 mM, the concentration of pectin was 2.5 mg / mL, the reaction temperature was 25°C, and the reaction time was 4 h, the particle size of SULMP-SeNPs was measured when the concentration of KI was 1, 5, 10, 15, and 20 mM, respectively. Figure 5 As shown, when the KI concentration is 15 mM, the particle size of the nano-selenium complex is the smallest.

[0065] Example 2: Verification Experiment

[0066] According to the preparation experiment of the low molecular weight pectin nano-selenium complex in Example 1, the optimal condition for the smallest particle size was optimized using the low molecular weight pectin concentration to obtain the best conditions. The experimental results are as follows: Figure 6 As shown in the figure, it can be seen that the optimal conditions are SULMP 2 mg / mL, Vc 35 mM, KI 15 mM, reaction at 30°C for 8 hours, and the particle size of the obtained SULMP-SeNPs is 110.1 nm.

[0067] According to the above optimal experimental conditions of Example 2, when pectin is replaced by SUPO or SP with the same concentration, pectin nano-selenium complexes SUPO-SeNPs or SP-SeNPs can be obtained respectively.

[0068] Example 3: Storage stability of low molecular weight pectin-nanoselenium complex

[0069] The three pectin nano-selenium complexes prepared under the optimal conditions of Example 2 were stored at 4°C, and their particle sizes were measured every 7 days using a laser particle size analyzer. Figure 7 It can be seen that the particle size of SULMP-SeNPs remained the most stable, with no significant changes within eight weeks, and the best storage stability.

[0070] Example 4: Characterization of Nano-Selenium Complex

[0071] The SULMP-SeNPs prepared under the optimal conditions of Example 2 were measured by scanning electron microscopy, infrared spectroscopy, and X-ray photoelectron spectroscopy.

[0072] 1. Scanning electron microscopy

[0073] Take an appropriate amount of freeze-dried SULMP-SeNPs powder sample and stick it on the conductive glue. Spray gold on the sample for 20 minutes, set the scanning voltage to 5kV and the magnification to 1.5k.

[0074] Figure 8 The microscopic morphology of SULMP-SeNPs can be observed. Through SEM, it can be seen that SULMP-SeNPs form spherical nanoparticles. After embedding nano-selenium, pectin changes from large flakes and particles to small fragments, and the smaller the particle size of SeNPs, the easier it is to agglomerate and form large particles. This shows that low molecular weight pectin does help form SeNPs with regular shapes and has the ability to prevent SeNPs from agglomerating.

[0075] 2. Infrared spectroscopy

[0076] The dried SULMP-SeNPs sample or SULMP sample was mixed with potassium bromide in a ratio of 1:100 and ground evenly. The mixture was then pressed into a sheet and scanned using an infrared spectrometer. The wave number range was 4000-400 cm -1 , scanning step length is 4.0cm -1

[0077] Fig. 9 There are no new absorption peaks in SULMP-SeNPs, indicating that no new bonds are formed. However, the -OH peak shows a peak from 3431 cm -1 To 3416cm -1 and 3423cm -1 The red shift indicates that the -OH of SULMP and the Se atoms of SeNPs form a strong bonding interaction (OH…Se) through a hydrogen-bond-like interaction. The results show that there is an interaction between the OH and CN groups of pectin oligosaccharides and Se atoms, and there is a strong electrostatic repulsion between the molecules, which prevents the aggregation of SeNPs particles and forms a stable spherical structure.

[0078] 3. X-ray photoelectron spectroscopy

[0079] X-ray photoelectron spectroscopy was used to perform full spectrum scans and fine spectrum scans on SULMP-SeNPs, and the fine spectrum was scanned at least 5 times.

[0080] Fig.10 (a) By performing XPS analysis on the surface of SULMP-SeNPs, we can clearly observe the presence of elements Se, O, and C. Fig.10(b) shows the Se3d spectrum of SULMP-SeNPs, and characteristic spin-orbit Se3d5 / 2 and Se3d3 / 2 can be observed after deconvolution. The binding energies of the Se3d peaks of SULMP-SeNPs were observed to be 55.5 eV, 55.6 eV, and 55.9 eV, respectively, indicating that Se in SULMP-SeNPs is in a zero-valent state. We found that the binding energy of the Se3d3 / 2 peak is 0.8 eV higher than that of the Se3d5 / 2 peak, with a spin-orbit splitting ratio of 2:3, and no peak was found at 59.5 eV, which confirms that Se(IV) is completely reduced to red elemental selenium (Se 0 ).

[0081] Example 5: Antibacterial activity assay

[0082] The three pectin nano-selenium complexes prepared under the optimal conditions of Example 2 were respectively subjected to antibacterial activity determination, and the selenium content was detected by inductively coupled plasma mass spectrometry (ICP-MS). Ultrapure water was used to prepare mother solutions with Se-SeNPs contents of 0.5, 1.0, 1.5, 2.0, and 2.5 mg / mL. The mother solutions were diluted to the test solution with the appropriate Se content according to the antibacterial effect, and sterilized by filtration with a water-based filter membrane. At the same time, they were compared with SP-SeNPs and SPUO-SeNPs. The bacterial suspension was diluted to 10 5 CFU / mL. Take 100 μL of the test solution and bacterial suspension into a 96-well plate, place it in a 37°C shaker and culture for 12 to 16 hours, and measure the OD of each well. 600 , add 100 μL of sterile ultrapure water as a positive control, add 100 μL of LB liquid culture medium as a negative control, and repeat each experiment 3 times. The inhibition rate is calculated using the following formula:

[0083]

[0084] Among them A 0 Indicates the absorbance of the sample replaced by ultrapure water, A s It indicates the absorbance measured after the test solution and bacterial suspension are mixed.

[0085] 1. Antibacterial effect on B. subtilis

[0086] Fig.11 The figure shows the comparison of the antibacterial effects of samples with different concentrations on B. subtilis. Compared with SP-SeNPs and SUPO-SeNPs, SULMP-SeNPs has the best antibacterial effect. When the selenium content of SULMP-SeNPs is 2.0 mg / mL, the antibacterial rate can reach 98%, which is much higher than other forms of embedded Se.

[0087] 2. Antibacterial effect on E.coli

[0088] Fig.12 The figure is a comparison of the antibacterial effects of samples of different concentrations on E. coli. Compared with SP-SeNPs and SUPO-SeNPs, SULMP-SeNPs has the best antibacterial effect. When the selenium content of SULMP-SeNPs is 1.2 mg / mL, the antibacterial rate can reach 98%, which is much higher than other forms of embedded Se.

[0089] 3. Antibacterial effect on S. aureus

[0090] Fig.13 The figure shows the comparison of the antibacterial effects of samples with different concentrations on S. aureus. Compared with SP-SeNPs and SUPO-SeNPs, SULMP-SeNPs have the best antibacterial effect. When the selenium content of SULMP-SeNPs is 75μg / mL, the antibacterial rate can reach 99%, which is much higher than other forms of embedded Se.

[0091] Example 6: Antitumor Activity Assay

[0092] 1. Inhibition of HepG2 tumor cells by sunflower low molecular weight pectin nano-selenium

[0093] First, culture cells by adding 1 mL of culture medium (DMEM medium for HepG2 cells and RPMI 1640 medium for L02 cells) and adjusting the cell concentration to 5 × 10 cells per mL by cell counting. 4 Then, 100 μL of cells were inoculated into a sterile 96-well plate and incubated at 37°C in a CO 2 The cells were cultured in an incubator for 24 h to ensure that the cells were fully attached.

[0094] The SP-SeNPs, SULMP-SeNPs, and SUPO-SeNPs solutions with Se contents of 5 μg / mL, 10 μg / mL, 15 μg / mL, 20 μg / mL, and 25 μg / mL were prepared by the dilution method. 1 μL of the prepared solution was added to a 96-well plate, and duplicate wells and zero wells were set. The 96-well plate with the added samples was placed in a 37°C CO 2 The cells were incubated in an incubator for 48 h. Afterwards, the samples and culture medium were discarded and 100 μL of the prepared CCK-8 solution (CCK-8 to culture medium volume ratio 1:10) was added. 2 Continue to incubate in the incubator for 1 to 3 hours. Measure the OD of the 96-well plate with an enzyme-labeled instrument. 450 The growth inhibition rate of HepG2 cells and the survival rate of normal L02 cells were calculated according to the following formula.

[0095]

[0096]

[0097] Where As is the absorbance of the experimental well, Ac is the absorbance of the control well (without adding samples), and Ab is the absorbance of the blank well (without adding cells and samples).

[0098] Fig.14 SP-SeNPs, SULMP-SeNPs and SUPO-SeNPs with different Se contents showed different inhibitory effects on HepG2 tumor cells, and the degree of inhibition was positively correlated with the Se content. SUPO-SeNPs with the smallest particle size had the weakest inhibitory effect on HepG2 tumor cells, which may be because Se inhibited tumor cells while SUPO promoted their growth, making the inhibitory effect weaker.

[0099] Fig.15 The toxicity of SP-SeNPs, SULMP-SeNPs and SUPO-SeNPs with different Se contents on L02 normal liver cells. These three types of nano-selenium can inhibit the growth of normal liver cells, but the degree of inhibition does not change with the change of Se content.

[0100] These results indicate that SeNPs encapsulated with SP, SULMP, and SUPO can enhance the inhibitory effect on HCC cells, but the selectivity between HCC cells and normal cells needs to be further improved.

[0101] 2. Production of reactive oxygen species in cells

[0102] Treat and collect cells according to the cytotoxicity assay method, and adjust the cell concentration to 5 × 10 per ml by cell counting. 4 After the cells attached to the wall, 1 μL of SP-SeNPs, SULMP-SeNPs, SUPO-SeNPs and their controls (cells without sample added) were added. The Se content and cytotoxicity were the same. The cells were placed in a 37°C CO 2 Culture in incubator for 48h

[0103] The ROS content in cells was detected using the reactive oxygen species detection kit from Beijing Solebow Technology Co., Ltd. The fluorescence intensity at an excitation wavelength of 488 nm and an emission wavelength of 525 nm was detected using a microplate reader, and the relative level of ROS release in HepG2 cells and L02 cells was calculated according to the following formula.

[0104]

[0105] A s : Fluorescence intensity of the sample well;

[0106] A 0 : Sunlight intensity in the control group.

[0107] Fig.16 , Fig.17 The effects of SP-SeNPs, SULMP-SeNPs and SUPO-SeNPs with different Se contents on the changes in ROS levels in HepG2 tumor cells and L02 normal liver cells were shown in Figure 2. When the Se content was 5-15 μg / mL, the ROS level of HepG2 tumor cells increased; when the Se content was 15-25 μg / mL, the ROS level of HepG2 tumor cells decreased, and the ROS level of SUPO-SeNPs>SULMP-SeNPs>SP-SeNPs. The experimental results showed that SP-SeNPs, SULMP-SeNPs and SUPO-SeNPs with different Se contents all led to an increase in the ROS level in HepG2 tumor cells and L02 normal cells. This suggests that SeNPs may inhibit the growth of HepG2 tumor cells and cause toxic reactions in L02 normal cells by increasing the ROS level.

[0108] It should be particularly noted that within the range of components, ratios and process parameters recorded in the present invention, other components, ratios or values ​​can be specifically selected to achieve the technical effects recorded in the present invention, so they will not be listed one by one.

[0109] The contents described in this specification are merely an enumeration of implementation forms of the inventive concept, and the protection scope of the present invention should not be regarded as being limited to the specific forms described in the embodiments.

Claims

1. A method for preparing a low molecular weight pectin nano-selenium complex, characterized in that The following steps are involved: 1) Preparation of low molecular weight pectin hydrolysate The pectin is dissolved in ultrapure water at a material-liquid mass ratio of 1:50-1:200, the pH is first adjusted to acidic, and then pectinase is added, and the temperature is maintained at 30-60°C for enzymatic hydrolysis. After the reaction is completed, the enzyme activity is immediately boiled to inactivate the enzyme, and the bottom precipitate is removed by centrifugation after cooling to room temperature, and the supernatant is collected as the enzymatic hydrolyzate; after the enzymatic hydrolyzate is filtered, 0.5-2% dry yeast by volume of the filtrate is added to the filtrate, and the fermentation is carried out at 25-35°C for 25-45 hours, and the fermentation is carried out by centrifugation, and the supernatant is collected to obtain the fermentation liquid; The fermentation broth was passed through a microfiltration device with a 30 kDa polyethersulfone membrane to remove the unhydrolyzed pectin, and then the small molecular weight polysaccharides were removed through a 10 kDa polyethersulfone membrane to obtain a low molecular weight pectin SULMP filtrate, which was then concentrated under reduced pressure using a rotary evaporator to obtain low molecular weight pectin SULMP, and the removed small molecular weight polysaccharides were recovered as pectin oligosaccharides SUPO; 2) Preparation of low molecular weight pectin nano-selenium complex The low molecular weight pectin SULMP in step 1) is mixed with potassium iodide, ascorbic acid and a tetravalent selenium source, dissolved in water to form a solution, stirred for reaction in a water bath, and the obtained reaction solution is dialyzed to obtain a low molecular weight pectin nano-selenium complex solution, which is then freeze-dried into a powdery solid to obtain a low molecular weight pectin nano-selenium complex SULMP-SeNPs.

2. The method for preparing a low molecular weight pectin nano-selenium complex as claimed in claim 1, characterized in that The pectinase is pectin lyase, and the enzyme activity concentration of the pectin lyase in the reaction solution of the enzymatic reaction is 5-15 U / mL, preferably 9-10 U / mL.

3. The method for preparing a low molecular weight pectin nano-selenium complex as claimed in claim 1, characterized in that In step 1), the pH is adjusted to 4.0-6.0, preferably 5.5, the reaction temperature is 30-60° C., preferably 45° C., and the enzymatic hydrolysis time is 2-8 h, preferably 3 h.

4. The method for preparing a low molecular weight pectin nano-selenium complex as claimed in claim 1, characterized in that In step 2), in the mixed solution for water bath reaction, the concentration of low molecular weight pectin is 1.5-3.5 mg / mL, preferably 2.5 mg / mL, the concentration of ascorbic acid is 20-40 mM, preferably 30-35 mM, and the molar concentration of potassium iodide is 1-20 mM, preferably 10-15 mM.

5. The method for preparing a low molecular weight pectin nano-selenium complex as claimed in claim 1, characterized in that Step 2) The reaction temperature is 25-35°C, preferably 30°C, the reaction time is 2-20h, preferably 8h, and the product needs to be left in the dark for 20-30h to age.

6. The method for preparing a low molecular weight pectin nano-selenium complex as claimed in claim 1, characterized in that In step 2), the dialysis process includes transferring the reaction solution to a 2500-3500Da dialysis bag, controlling the dialysis temperature at 2-6°C in a light-proof environment, and continuing the dialysis for 2-4 days, preferably 3 days.

7. The method for preparing a low molecular weight pectin nano-selenium complex as claimed in claim 1, characterized in that In step 2), the low molecular weight pectin nano-selenium complex solution is placed in a -20°C ± 2°C refrigerator for pre-freezing for 10-12 hours, and then the pre-frozen low molecular weight pectin nano-selenium complex is placed in a freeze dryer at -45 to -55°C for freeze drying for 30-40 hours, and the vacuum degree of the freeze dryer is above 0.09MPa.

8. A low molecular weight pectin nano-selenium complex prepared by the method according to any one of claims 1 to 7.

9. Use of the low molecular weight pectin nano-selenium complex as claimed in claim 8 in the preparation of antibacterial or anti-tumor drugs.

Images