CN117257832A - Ganoderan selenium nanoparticle and preparation method thereof - Google Patents
Ganoderan selenium nanoparticle and preparation method thereof Download PDFInfo
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- 239000011669 selenium Substances 0.000 title claims abstract description 96
- 229910052711 selenium Inorganic materials 0.000 title claims abstract description 74
- BUGBHKTXTAQXES-UHFFFAOYSA-N Selenium Chemical compound [Se] BUGBHKTXTAQXES-UHFFFAOYSA-N 0.000 title claims abstract description 72
- 239000002105 nanoparticle Substances 0.000 title claims abstract description 70
- 238000002360 preparation method Methods 0.000 title claims abstract description 21
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- RJFAYQIBOAGBLC-UHFFFAOYSA-N Selenomethionine Natural products C[Se]CCC(N)C(O)=O RJFAYQIBOAGBLC-UHFFFAOYSA-N 0.000 description 1
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- MGJZITXUQXWAKY-UHFFFAOYSA-N diphenyl-(2,4,6-trinitrophenyl)iminoazanium Chemical compound [O-][N+](=O)C1=CC([N+](=O)[O-])=CC([N+]([O-])=O)=C1N=[N+](C=1C=CC=CC=1)C1=CC=CC=C1 MGJZITXUQXWAKY-UHFFFAOYSA-N 0.000 description 1
- BVTBRVFYZUCAKH-UHFFFAOYSA-L disodium selenite Chemical compound [Na+].[Na+].[O-][Se]([O-])=O BVTBRVFYZUCAKH-UHFFFAOYSA-L 0.000 description 1
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- GRVFOGOEDUUMBP-UHFFFAOYSA-N sodium sulfide (anhydrous) Chemical compound [Na+].[Na+].[S-2] GRVFOGOEDUUMBP-UHFFFAOYSA-N 0.000 description 1
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- DRDVZXDWVBGGMH-UHFFFAOYSA-N zinc;sulfide Chemical compound [S-2].[Zn+2] DRDVZXDWVBGGMH-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- 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
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
-
- 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
- A61K33/04—Sulfur, selenium or tellurium; Compounds thereof
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P39/00—General protective or antinoxious agents
- A61P39/06—Free radical scavengers or antioxidants
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B82—NANOTECHNOLOGY
- B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
- B82Y40/00—Manufacture or treatment of nanostructures
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B82—NANOTECHNOLOGY
- B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
- B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/04—Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/14—Preparation of compounds containing saccharide radicals produced by the action of a carbohydrase (EC 3.2.x), e.g. by alpha-amylase, e.g. by cellulase, hemicellulase
Abstract
The invention provides ganoderma lucidum polysaccharide selenium nano-particles and a preparation method thereof. Degreasing and depigmenting ganoderma lucidum spore powder by using 95% ethanol, and volatilizing; adding distilled water into the volatilized ganoderma lucidum spore powder according to a certain feed liquid ratio, performing ultrasonic extraction, extracting the extracting solution in a water bath, repeatedly extracting, combining the filtrates, and concentrating to obtain a concentrated solution; sequentially adding dehydrated pyridine and beta-galactosidase solution into the concentrated solution for reaction, and freeze-drying the supernatant after the reaction to obtain the ganoderma lucidum polysaccharide with small molecular weight; then adding the small molecular weight ganoderan into selenium oxychloride solution, oscillating at low speed until light red color is produced; and then rapidly oscillating until the red color disappears to form a stable ganoderan selenium nanoparticle solution, and freeze-drying to obtain ganoderan selenium nanoparticles. The ganoderan nanoparticle obtained by the invention has good stability, strong antioxidation capability and remarkable inhibition activity on HepG2 cells and A549 cells.
Description
Technical Field
The invention relates to the field of biological new materials, in particular to ganoderma lucidum polysaccharide selenium nano-particles and a preparation method thereof.
Background
Ganoderma lucidum has a medicinal history of over 2000 in China and is regarded as a magic article for nourishing, strengthening and consolidating body resistance by the medical staff of the past generation. The spore powder has effects of rapidly improving immunity, lowering blood pressure, lowering blood sugar, reducing cholesterol, improving microcirculation, preventing cardiovascular and cerebrovascular diseases, calming heart, tranquilizing mind, promoting sleep, invigorating spleen, stimulating appetite, relieving cough and asthma, caring skin, prolonging life, relieving fatigue, and strengthening body resistance. In particular, triterpenes and nucleoside compounds in the spore powder can destroy telomerase specific to malignant tumor cells, inhibit the growth of immortalized and mutated cells, and cut off the DNA chain of tumor cells to lose the growth capacity until apoptosis. The spore powder also has the function of resisting radiation, can relieve or eliminate leucopenia caused by radiotherapy and chemotherapy, prevent cancer cell metastasis, eliminate specific severe pain of cancer, improve treatment tolerance, promote appetite, shrink tumor and prevent postoperative recurrence of cancer.
Polysaccharides are natural polymers made from more than 10 monosaccharides linked by one or more glycosidic linkages. Natural or modified polysaccharides contain high levels of functional groups, including hydroxyl, amino, sulfate, and carboxylic acid groups, facilitating the formation of intermolecular and intramolecular hydrogen bonds along the polymer chain. The application of nanotechnology in the food industry has shown great potential in preventing contamination, improving flavor and texture, extending the shelf life of foods and improving food quality. Polymer nanoparticles based on biocompatible and biodegradable polymers have attracted attention in recent years. Among these biologically active nanocomposites, polysaccharides are considered to be the most promising source of nanoparticle production because of their relative stability and cost effectiveness. Therefore, polysaccharides can be used as ideal templates for nanoparticle synthesis using modern nanotechnology.
Selenium is one of the essential trace nutrient elements for human body, and reasonable intake of selenium element has important effects of keeping healthy and preventing diseases. Research shows that selenium enters human body to participate in the synthesis of in vivo antioxidant enzyme system (such as glutathione catalase synthesis), and can rapidly remove in vivo free radicals, thereby effectively defending the damage of free radicals to cells. Selenium deficiency can cause various diseases such as cancer, heart disease, keshan disease, immune system dysfunction and the like, while selenium belongs to a powerful medicine in cancer medicine prevention, and selenium supplementing can effectively reduce the incidence of cancer, and is called as anticancer king. Currently, selenium supplementation is often added to many food supplements in the form of sodium selenite, sodium selenate, selenomethionine. However, numerous studies have shown that the range between effective and toxic doses of selenium is extremely narrow, which greatly limits the use of selenium in food supplements. Nano selenium (SeNPs) has the characteristic of high bioavailability, and has acute, sub-chronic and long-term toxicity lower than inorganic selenium and organic selenium. SeNPs have been demonstrated to exert anticancer, antioxidant, etc., but their efficacy is based on the stabilization of the template, that is, seNPs without template are extremely susceptible to aggregation and inactivation and are not active, which has become a bottleneck limiting the application of SeNPs. The addition of the template with biological activity can not only stabilize the Senps, but also synergistically enhance the health efficacy of the Senps. The research at home and abroad shows that the polysaccharide can be used as a template to stabilize the SenPs. Ganoderan is used as one of main active ingredients of ganoderma lucidum, has been proved to have various biological activities of resisting cancer, resisting oxidation, protecting liver, regulating immunity and the like, and has the potential of synergistically enhancing the functional activity of SeNPs besides the stabilizing effect on SeNPs. Therefore, searching for a template with biological activity, developing a nano selenium preparation with strong activity, low toxicity and good stability is an important research direction in the related fields at home and abroad. The literature reports that polysaccharide with high molecular weight is used as a template to construct nano particles, but the nano particles constructed by polysaccharide with lower molecular weight have relatively simple structure, poor stability and low bioavailability, which always plagues the further utilization of ganoderan.
Several methods for preparing nano-selenium have been patented by the state of the art (grant publication number CN 101544359 A;CN 102228469 B;CN 103420344A). Research shows that the bioavailability and antitumor activity of selenium are greatly different along with the change of nano-scale, and the synthesized surface is not easy to aggregate and precipitate, which has become the bottleneck for limiting the application of the SENPs. Therefore, the preparation of the Senps with small particle size and narrow distribution, and the functional modification of the surface by using polysaccharide, so as to obtain the selenium nano particles with good stability, become the hot spot of the current research.
Therefore, the invention designs the ganoderan nanoparticle with strong development activity, low toxicity and good stability and the preparation method thereof.
Disclosure of Invention
The invention provides ganoderma lucidum polysaccharide selenium nano-particles and a preparation method thereof, and aims to solve the problems existing in the background technology.
In order to achieve the above purpose, the embodiment of the invention provides a ganoderan selenium nanoparticle and a preparation method thereof, wherein the method is to defat and depigment ganoderma lucidum spore powder with 95% ethanol and volatilize; adding distilled water into the volatilized ganoderma lucidum spore powder according to a certain feed liquid ratio, performing ultrasonic extraction, extracting the extracting solution in a water bath, repeatedly extracting, combining the filtrates, and concentrating to obtain a concentrated solution; sequentially adding dehydrated pyridine and beta-galactosidase solution into the concentrated solution for reaction, and freeze-drying the supernatant after the reaction to obtain the ganoderma lucidum polysaccharide with small molecular weight; then adding the small molecular weight ganoderan into selenium oxychloride solution, oscillating at low speed until light red color is produced; and then rapidly oscillating until the red color disappears to form a stable ganoderan selenium nanoparticle solution, and freeze-drying to obtain ganoderan selenium nanoparticles. The ganoderan nanoparticle obtained by the invention has good stability, strong antioxidation capability and remarkable inhibition activity on HepG2 cells and A549 cells.
One aspect of the invention provides a method for preparing ganoderan selenium nanoparticles, comprising the following steps:
s1: pretreatment of ganoderma lucidum spore powder:
degreasing and depigmenting Ganoderma spore powder with 95% ethanol, and volatilizing; adding distilled water into the volatilized ganoderma lucidum spore powder according to a certain feed liquid ratio, performing ultrasonic extraction, extracting the extracting solution in a water bath, repeatedly extracting, combining the filtrates, and concentrating to obtain a concentrated solution;
s2: preparation of small molecular weight ganoderan:
sequentially adding dehydrated pyridine and beta-galactosidase solution into the concentrated solution for reaction, and freeze-drying the supernatant after the reaction to obtain the ganoderma lucidum polysaccharide with small molecular weight;
s3: adding the small molecular weight ganoderan into selenium oxychloride solution, and oscillating at low speed until light red color is generated; and then rapidly oscillating until the red color disappears to form a stable ganoderan selenium nanoparticle solution, and freeze-drying to obtain ganoderan selenium nanoparticles.
Preferably, the ganoderma lucidum spore powder is added with distilled water according to a feed liquid ratio of 1:15-25 g/mL.
Preferably, in the step S1, ultrasonic extraction is carried out at the temperature of 45-55 ℃ with the ultrasonic power of 100-200W; the water bath temperature is 75-85 ℃; concentrating the filtrate to 1:5, wherein the concentration temperature is 60-65 ℃ and the pressure is 0.08-0.09MPa.
Preferably, in the step S1, the degreasing and depigmenting temperature is 70-80 ℃, the time is 3 hours, and the repetition times are 2 times; the extraction time is 30min, and the repetition time is 2 times.
Preferably, the ratio of the dehydrated pyridine to the beta-galactosidase solution in the step S2 is 1:1-3; the concentration of the dehydrated pyridine and beta-galactosidase solution is 1-2%.
Preferably, the reaction temperature in the step S2 is 4 ℃, and the reaction time is 24-30 hours; the ganoderan with small molecular weight is 10-12 k dal.
Preferably, the selenium oxychloride solution in step S3 is 1l 0.1%. More preferably, the ratio of selenium to small molecular weight ganoderan in ganoderan selenium nanoparticles is 1:3.
Preferably, the low-speed oscillation condition in the step S3 is 4 ℃, 20-40 rpm, 3h; the rapid oscillation is at 25 ℃ and 160-200 rpm for 7h. More preferably, the reaction is complete by shaking for 4 hours at a temperature of 25℃and a shaking speed of 130 rpm.
Preferably, the low temperature shaking table is used for replacing the traditional magnetic stirring oscillation in the steps S2 and S3.
Based on the general inventive concept, the invention also provides the ganoderma lucidum polysaccharide selenium nanoparticle prepared by the preparation method, and the particle size of the ganoderma lucidum polysaccharide selenium nanoparticle is 140-150 nm. More preferably, the particle size is 146nm.
Reaction mechanism
Beta-galactosidase can directionally hydrolyze beta-glycosidic bond of ganoderan, and reduce branched chains of ganoderan, thereby directionally reducing molecular weight of products, but due to high efficiency of beta-galactosidase, the space structure of ganoderan is easily destroyed at too high speed of enzymolysis, and more linear structures are formed. The dehydrated pyridine can reduce the effective moisture in a reaction system, reduce the enzymolysis speed, form a slow reaction system, stop the reaction when appropriate, obtain more small molecular weight polysaccharide, release selenium particles timely after selenium oxychloride is added, enter space sites of ganoderan timely, stabilize the space structure of the small molecular weight ganoderan while protecting beta-glycosidic bond, and convert the space structure into ganoderan nano-selenium particles.
The scheme of the invention has the following beneficial effects:
(1) Compared with the traditional prepared polysaccharide nanoparticle, the ganoderan selenium nanoparticle has the advantages of smaller particle size and easy absorption, and the ganoderan selenium nanoparticle is beneficial to the low-molecular ganoderan with higher hydroxyl ratio, so that the ganoderan selenium nanoparticle has smaller particle size and is easier to absorb.
(2) The ganoderan selenium nanoparticle promotes anticancer activity and antioxidant activity; the advantage of high hydroxyl number of the low molecular ganoderan is utilized to stabilize the selenium nano-particles, and the free hydroxyl groups are promoted to combine with the nano-particles to form hydrogen bonds, so that the nano-particles have good stability and dispersibility, and a synergistic effect is achieved between the two; and the ganoderan stable nanoparticles can be stably stored for 90 days at 4 ℃.
(3) The invention adopts the dehydrated pyridine to ensure the yield of the ganoderan with small molecular weight (the contact between enzyme and glycosidic bond is increased by limiting free water) and promote the combination of ganoderan and nano particles (by activating selenium particles).
(4) The preparation method of the low-temperature shaking table has the advantages of mild and controllable reaction conditions and capability of realizing large-scale production.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a transmission electron microscope image of GLP-SeNPs (1/3) and SeNPs and EDX analysis of GLP-SeNPs (1/3) of an embodiment of the invention; wherein FIG. 1A is a transmission electron microscope image of 1/3GLP-SeNPs in the absence of GLP (A), and FIG. 1B is a transmission electron microscope image of Se particles in the presence of GLP (B); FIG. 1C is an EDX analysis of GLP-SeNPs (1/3);
FIG. 2 is a graph showing the particle size distribution of GLP-SeNPs (1/3) according to an embodiment of the present invention;
FIG. 3 is a UV-Vis absorption spectrum of GLP-SeNPs solutions of this invention at four different Se/GLP ratios (1/20, 1/3, 1/1 and 4/3);
FIG. 4 is FT-IR spectra of GLP-SeNPs of pure GLP and different Se/P ratios of an embodiment of the invention;
FIG. 5 is a graph showing the stability of selenium nanoparticle of ganoderan according to an embodiment of the present invention; wherein, FIG. 5A is the solution before storage, FIG. 5B is the solution after storage for 30d at 4 ℃, and FIG. 5C is the potential results of different Se/GLP;
FIG. 6 shows the free radical scavenging ability of the ganoderan selenium nanoparticle of an embodiment of the present invention; wherein, FIG. 6A is DPPH radical scavenging rate and FIG. 6B is PTIO radical scavenging rate;
FIG. 7 inhibition of A549 cells (A) HepG2 cells (B) 293T cells (C) by GLP and GLP-SeNPs of the examples of the invention;
FIG. 8 is a scanning electron micrograph of GLP1-SeNPs of an embodiment of this invention.
Detailed Description
In order to make the technical problems, technical solutions and advantages to be solved more apparent, the following detailed description will be given with reference to the accompanying drawings and specific embodiments.
Unless defined otherwise, all technical and scientific terms used hereinafter have the same meaning as commonly understood by one of ordinary skill in the art. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the scope of the present invention.
Unless otherwise specifically indicated, the various raw materials, reagents, instruments, equipment and the like used in the present invention are commercially available or may be prepared by existing methods.
The invention provides ganoderma lucidum polysaccharide selenium nano-particles and a preparation method thereof, aiming at the existing problems.
Example 1
A preparation method of ganoderma lucidum polysaccharide selenium nano-particles comprises the following steps:
(1) Pretreatment of ganoderma lucidum spore powder:
defatting Ganoderma spore powder with 95% ethanol, removing pigment, and volatilizing. The ratio of the material to the liquid is 1:20g/mL, adding distilled water with power of 150W, performing ultrasonic extraction at 50deg.C, extracting the extractive solution in water bath at 80deg.C, repeatedly extracting, mixing filtrates, and concentrating to 1:5.
(2) Preparation of small molecular weight ganoderan:
250mL of dehydrated pyridine and beta-galactosidase solution are sequentially added into the concentrated solution, the temperature is kept at 4 ℃ for 27+/-2 hours, and the supernatant is taken and freeze-dried to obtain the ganoderan with the molecular weight of 12 kdal.
(3) Adding the prepared ganoderan into 1L of 0.1% selenium oxychloride solution, and oscillating at low speed at 4 ℃ on a shaking table at 30+ -5 rpm until pale red is generated;
(4) And (3) rapidly oscillating at 180+/-10 rpm for 3 hours at 25 ℃ until the red color disappears, forming a stable ganoderan nanoparticle solution, and freeze-drying to obtain ganoderan selenium nanoparticles.
Example 2
A preparation method of ganoderma lucidum polysaccharide selenium nano-particles comprises the following steps:
(1) Pretreatment of ganoderma lucidum spore powder:
defatting Ganoderma spore powder with 95% ethanol, removing pigment, and volatilizing. The ratio of the material to the liquid is 1:15g/mL, adding distilled water with the power of 100W, performing ultrasonic extraction at 45 ℃, extracting the extracting solution in a water bath at 75 ℃, repeatedly extracting, combining the filtrates, and concentrating to 1:5.
(2) Preparation of small molecular weight ganoderan:
250mL of dehydrated pyridine and beta-galactosidase solution are sequentially added into the concentrated solution, the temperature is kept at 4 ℃ for 25+/-1 h, and the supernatant is taken and freeze-dried to obtain the ganoderan with the molecular weight of 10 kdal.
(3) Adding the prepared ganoderan into 1L of 0.1% selenium oxychloride solution, and oscillating at low speed at 4 ℃ on a shaker at 35+/-5 rpm to generate pale red;
(4) Rapidly oscillating at 170+ -10 rpm at 25deg.C for 3 hr until red color disappears to form stable ganoderan nanoparticle solution, and lyophilizing to obtain ganoderan selenium nanoparticle.
Comparative example 1
A preparation method of ganoderma lucidum polysaccharide selenium nano-particles comprises the following steps:
(1) The ganoderan was obtained as in example 1.
(2) 2mmol of zinc acetate are poured into 5ml of water at room temperature. Then, the ganoderan solution is added to the mixture and stirred on a magnetic stirrer for 30min.
(3) Meanwhile, adding sodium sulfide solution into zinc acetate and ganoderan solution to generate white zinc sulfide particles. After the reaction is finished, the prepared nano-carrier is dispersed in ice bath, and a probe type ultrasonic homogenizer is used for homogenizing for 20min, so that the zinc sulfide-ganoderan hybrid nano-particles are homogenized. And then freeze-drying the obtained solution to obtain the ganoderma lucidum zinc nano-particles.
Characterization experiments
1. Cell culture and antitumor ability assay
Placing HepG2 cells and A549 cells in CO 2 Incubator (37 ℃,5% CO) 2 ) Culturing to a density of about 30%. 100 μl of cells were plated in 96-well plates, 6 parallel wells were plated, and a blank was set up. After cell adhesion growth, GLP-SeNPs samples with different concentrations (5, 25, 50, 100 and 200 mu g/mL) are added, the culture is carried out for 48 hours, and the condition of inhibition of sample treatment with different concentrations on HepG2 cells and A549 cells is detected by using a cck8 method. Adding 100 mu g/mL of cck8 solution into 100 mu L, incubating for 30min, measuring an OD value at 450nm by using an enzyme label instrument, and detecting the absorbance of cells, wherein the calculation formula is as follows:
cell proliferation inhibition ratio = 1-a Sample /A Control ×100％。
2. DPPH and PTIO radical scavenging ability
1mgDPPH/PTIO is weighed and dissolved in 10mL absolute ethyl alcohol to be prepared for use. The samples and positive control were then first prepared as 1mg/mL stock solutions, and diluted in a gradient to 5, 10, 20, 40, 60, 80, 100. Mu.g/mL solutions. In a 96-well plate, 100. Mu.L of DPPH/PTIO solution was added with 100. Mu.L of the sample solution, reacted in a dark place for 30min, and absorbance was measured at 517 nm. The calculation formula is as follows:
clearance = (1-a Sample /A Control )*100％
FIGS. 1A and 1B are transmission electron microscope images of GLP-SepPs (1/3) and SepPs, respectively. FIG. 1C is an EDX (energy dispersive X-ray spectrometer) of GLP-SeNPs (1/3). As shown in the results, seNPs are aggregated rod-like crystals Se (FIG. 1A), while GLP-SeNPs (1/3) are dispersed spheres (FIG. 1B). It is shown that GLP-SeNPs (1/3) prevent zero-valent selenium aggregation through polysaccharide, and the formed GLP-SeNPs have good dispersibility. Whereas GLP-SeNPs (1/3) had a selenium content of 16% (FIG. 1C).
FIG. 2 is a graph of GLP-SeNPs (1/3) particle size distribution, showing that Se/P (1/1) has a minimum average particle size of 138nm (Table 1), se/P (1/3) has the largest particle size of 146nm, and PDI value of 0.268 < 0.7, within the application range of the theoretical model of the nanoparticle analyzer (FIG. 2). As the Se/GLP ratio increases, the particle size becomes smaller, but reaches the minimum when increasing to 1/1, and the Se/GLP ratio is continuously increased, so that the particle size is increased.
TABLE 1 average particle size distribution of GLP-SeNPs of four Se/GLPs
FIG. 3 is a UV-Vis absorption spectrum of GLP-SeNPs solutions at four different Se/GLP ratios (1/20, 1/3, 1/1 and 4/3). The results show that GLPS-SepPs with different Se/GLP ratios have a wider absorption peak at 200nm-600nm due to the formation of SepPs, which causes plasmon resonance (LSPR) to exist on the particle surface. And as the Se/GLP ratio increases, the peak at 260nm is higher, whereas when the Se/P ratio exceeds 1/1, the peak intensity does not increase any more (FIG. 3).
FIG. 4 is FT-IR spectra of GLP-senPs of pure GLP and different Se/P ratios. FIG. 4 shows FT-IR spectra of pure GLPS and GLPS-SeNPs of different Se/P ratios. The characteristic peaks of the GLPS-SeNPs spectrum patterns are similar to those of the polysaccharide, which indicates that the polysaccharide exists in the nano material. And at 3300-3450cm -1 The strong peak at which is attributable to OH stretching vibration, while the weak peak at 2922cm-1 indicates CH stretching vibration of the polysaccharide. 1000-1200cm -1 The band of (2) is assigned to carbonyl groups. Whereas the-OH ratio in GLPS-SeNPs is from 3388.6cm compared to pure GLP -1 Offset to 3376cm -1 Nearby, it was shown that free hydroxyl groups in GLP were reduced after Se binding. And the imino group at 1649 is also offset, indicating that the hydroxyl and imino groups in GLP may interact with the selenium atom.
FIG. 5A shows the potential results of a pre-storage solution, B a solution after 30d storage at 4℃and C different Se/GLP. The results show that, in FIG. 5A, there are four proportions of nanoparticles before storage, the Sepps as control, there was little precipitation in red before storage, the solution was colorless after storage, and there was a large amount of precipitation in the bottom (FIG. 5B). The Se/GLPs (1/20) and the Se/GLPs (1/3) have no obvious change before and after storage, the Se/GLPs (1/1) solution becomes turbid, but no precipitate is separated out, the Se/GLPs (4/3) solution has light color, and a little precipitate exists at the bottom. Se/GLPs (4/3) may be due to the presence of significant amounts of SenPs, causing particle collisions and precipitation. The other three groups were all stable for 30d at 4 ℃. Furthermore, at Se/GLP of 1/3, the potential was-31 mV, and the colloidal stability was significantly better than that of the other three groups (FIG. 5C). In conclusion, the selenium nanoparticle constructed at Se/GLP of 1/3 has the best stability and particle size of 146nm.
FIG. 6ADPPH radical scavenging rate B PTIO radical scavenging rate. The results show that the clearance effect of GLP-SeNPs with the same concentration on DPPH/PTIO is stronger than that of polysaccharide, but weaker than Vc.
FIG. 7 inhibition of A549 cells (A) HepG2 cells (B) 293T cells (C) by GLP and GLP-SeNPs. The results show that GLP-SeNPs remarkably inhibit proliferation of human lung cancer cells (A549 cells) and human liver cancer cells (HepG 2 cells) and have no toxic effect on human normal cells (293T cells). Indicating that it has strong anticancer activity.
FIG. 8 scanning electron micrograph of GLP 1-SeNPs. The result shows that the prepared selenium nano-particles have large particle size and poor dispersibility.
In conclusion, the influence of ganoderan with different molecular weights on selenium nanoparticles, the influence of polysaccharide with different mass ratios and selenium on the selenium nanoparticles are compared, and the influence of constant temperature shaking tables with different temperature and rotating speeds to replace magnetic stirring is compared. The result shows that the optimal selenium-polysaccharide ratio of the selenium nano-particles prepared from the ganoderma lucidum polysaccharide with small molecular weight is 1:3; and the shaking for 4 hours can thoroughly react when the temperature of the constant-temperature shaking table is 25 ℃ and the rotating speed is 130rpm, so that the selenium nano-particles with the particle size of 146nm can be obtained, and can be stored for at least 30 days at the temperature of 4 ℃, and the ganoderan selenium nano-particles have good antioxidant capacity and anticancer activity.
While the foregoing is directed to the preferred embodiments of the present invention, it will be appreciated by those skilled in the art that various modifications and adaptations can be made without departing from the principles of the present invention, and such modifications and adaptations are intended to be comprehended within the scope of the present invention.
Claims (10)
1. The preparation method of the ganoderan selenium nanoparticle is characterized by comprising the following steps:
s1: pretreatment of ganoderma lucidum spore powder:
degreasing and depigmenting Ganoderma spore powder with 95% ethanol, and volatilizing; adding distilled water into the volatilized ganoderma lucidum spore powder according to a certain feed liquid ratio, performing ultrasonic extraction, extracting the extracting solution in a water bath, repeatedly extracting, combining the filtrates, and concentrating to obtain a concentrated solution;
s2: preparation of small molecular weight ganoderan:
sequentially adding dehydrated pyridine and beta-galactosidase solution into the concentrated solution for reaction, and freeze-drying the supernatant after the reaction to obtain the ganoderma lucidum polysaccharide with small molecular weight;
s3: adding the small molecular weight ganoderan into selenium oxychloride solution, and oscillating at low speed until light red color is generated; and then rapidly oscillating until the red color disappears to form a stable ganoderan selenium nanoparticle solution, and freeze-drying to obtain ganoderan selenium nanoparticles.
2. The method for preparing the ganoderan selenium nanoparticle according to claim 1, wherein the ganoderan spore powder is added with distilled water according to a feed liquid ratio of 1:15-25 g/mL.
3. The method for preparing the ganoderan selenium nanoparticle according to claim 2, wherein the ultrasonic power is set to be 100-200W and the ultrasonic extraction is performed at 45-55 ℃ in the step S1; the water bath temperature is 75-85 ℃; concentrating the filtrate to 1:5, wherein the concentration temperature is 60-65 ℃ and the pressure is 0.08-0.09MPa.
4. The method for preparing the ganoderan selenium nanoparticle according to claim 3, wherein the degreasing and depigmenting temperature in the step S1 is 70-80 ℃, the time is 3 hours, and the repetition number is 2 times; the extraction time is 30min, and the repetition time is 2 times.
5. The method for preparing the ganoderan selenium nanoparticle according to claim 4, wherein the ratio of the dehydrated pyridine to the beta-galactosidase solution in the step S2 is 1:1-3; the concentration of the dehydrated pyridine and beta-galactosidase solution is 1-2%.
6. The method for preparing the ganoderan selenium nanoparticle according to claim 5, wherein the reaction temperature in the step S2 is 4 ℃ and the reaction time is 24-30 hours; the ganoderan with small molecular weight is 10-12 k dal.
7. The method for preparing ganoderan selenium nanoparticle according to claim 6, wherein the selenium oxychloride solution in step S3 is 1l of 0.1%; the ratio of selenium to small molecular weight ganoderan in ganoderan selenium nanoparticle is 1:3.
8. The method for preparing selenium nanoparticle of ganoderan according to claim 7, wherein the low-speed oscillation condition in the step S3 is 4 ℃, 20-40 rpm, 3h; the rapid oscillation is at 25 ℃ and 160-200 rpm for 7h.
9. The method for preparing selenium nanoparticle of ganoderan according to claim 8, wherein the steps S2 and S3 are performed by shaking with shaking table.
10. The ganoderan selenium nanoparticle prepared by the preparation method of any of claims 1-9, wherein the ganoderan selenium nanoparticle has a particle size of 140-150 nm.
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