CN113875989B - Functionalized water-soluble nano-selenium, preparation method and application - Google Patents
Functionalized water-soluble nano-selenium, preparation method and application Download PDFInfo
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- CN113875989B CN113875989B CN202111113697.XA CN202111113697A CN113875989B CN 113875989 B CN113875989 B CN 113875989B CN 202111113697 A CN202111113697 A CN 202111113697A CN 113875989 B CN113875989 B CN 113875989B
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- 229910052711 selenium Inorganic materials 0.000 title claims abstract description 54
- 239000011669 selenium Substances 0.000 title claims abstract description 54
- 238000002360 preparation method Methods 0.000 title claims abstract description 19
- 229940091258 selenium supplement Drugs 0.000 claims abstract description 53
- 238000000855 fermentation Methods 0.000 claims abstract description 51
- 230000004151 fermentation Effects 0.000 claims abstract description 51
- 238000012258 culturing Methods 0.000 claims abstract description 45
- 241001486992 Taiwanofungus camphoratus Species 0.000 claims abstract description 42
- 238000003756 stirring Methods 0.000 claims abstract description 41
- 239000000243 solution Substances 0.000 claims abstract description 35
- BVTBRVFYZUCAKH-UHFFFAOYSA-L disodium selenite Chemical compound [Na+].[Na+].[O-][Se]([O-])=O BVTBRVFYZUCAKH-UHFFFAOYSA-L 0.000 claims abstract description 31
- 235000015921 sodium selenite Nutrition 0.000 claims abstract description 31
- 239000011781 sodium selenite Substances 0.000 claims abstract description 31
- 229960001471 sodium selenite Drugs 0.000 claims abstract description 31
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 claims abstract description 26
- BUGBHKTXTAQXES-UHFFFAOYSA-N Selenium Chemical compound [Se] BUGBHKTXTAQXES-UHFFFAOYSA-N 0.000 claims abstract description 22
- 239000000284 extract Substances 0.000 claims abstract description 22
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims abstract description 21
- 206010038389 Renal cancer Diseases 0.000 claims abstract description 18
- 238000010438 heat treatment Methods 0.000 claims abstract description 18
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- 238000006243 chemical reaction Methods 0.000 claims abstract description 16
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- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 36
- 244000061456 Solanum tuberosum Species 0.000 description 30
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- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 description 6
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- 210000003734 kidney Anatomy 0.000 description 4
- GBMDVOWEEQVZKZ-UHFFFAOYSA-N methanol;hydrate Chemical compound O.OC GBMDVOWEEQVZKZ-UHFFFAOYSA-N 0.000 description 4
- 239000000126 substance Substances 0.000 description 4
- 206010028980 Neoplasm Diseases 0.000 description 3
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- 229930003316 Vitamin D Natural products 0.000 description 1
- QYSXJUFSXHHAJI-XFEUOLMDSA-N Vitamin D3 Natural products C1(/[C@@H]2CC[C@@H]([C@]2(CCC1)C)[C@H](C)CCCC(C)C)=C/C=C1\C[C@@H](O)CCC1=C QYSXJUFSXHHAJI-XFEUOLMDSA-N 0.000 description 1
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- KBZOIRJILGZLEJ-LGYYRGKSSA-N argipressin Chemical compound C([C@H]1C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CSSC[C@@H](C(N[C@@H](CC=2C=CC(O)=CC=2)C(=O)N1)=O)N)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCCN=C(N)N)C(=O)NCC(N)=O)C1=CC=CC=C1 KBZOIRJILGZLEJ-LGYYRGKSSA-N 0.000 description 1
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- 230000033764 rhythmic process Effects 0.000 description 1
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- 150000003710 vitamin D derivatives Chemical class 0.000 description 1
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/16—Inorganic salts, minerals or trace elements
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- A23L31/00—Edible extracts or preparations of fungi; Preparation or treatment thereof
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- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K33/00—Medicinal preparations containing inorganic active ingredients
- A61K33/04—Sulfur, selenium or tellurium; Compounds thereof
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- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/06—Fungi, e.g. yeasts
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
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Abstract
The invention discloses a functional water-soluble nano selenium, a preparation method and application thereof, which are characterized in that radix pseudostellariae is taken as a raw material, and is extracted by methanol aqueous solution to obtain radix pseudostellariae extract; culturing Antrodia camphorata strains in a fermentation medium containing sodium selenite to obtain Antrodia camphorata mycelium fermentation broth; then adding sodium selenite solution into Antrodia camphorate mycelium fermentation broth, stirring, mixing, slowly adding ascorbic acid solution, stirring, heating for reaction, slowly dripping radix Pseudostellariae extractive solution during reaction, dialyzing after reaction, and lyophilizing. The nano selenium has good water solubility, and can be used for preparing health products or medicines for preventing and treating renal cancer after functional modification.
Description
Technical Field
The invention relates to nano-selenium, in particular to functionalized water-soluble nano-selenium, a preparation method and application thereof. Belongs to the technical field of nano selenium preparation.
Background
The kidney is located behind the peritoneal cavity of the human waist, including left and right kidneys, and plays an important role in the circulation and excretion of humans, mainly comprising: discharging waste and medicines in the body; maintaining the balance of water, pH value and electrolyte in the body; producing vasopressin to control blood pressure; vitamin D production to control calcium absorption and bone development; erythropoiesis is produced to maintain normal metabolism of the red blood cells.
Since the kidney is an indispensable organ in the human body, when the kidney function starts to be abnormal, the regulatory efficacy of the kidney will be greatly reduced, except that wastes, drugs and metabolites in the human body cannot be normally discharged out of the body; more serious, when kidney function is severely abnormal, uremia is also caused to endanger the life of the patient. Renal cancer is a highly malignant tumor in the urinary system, and it is feared that renal cancer cells develop slowly in an early stage, so that early symptoms are not developed, and pain and hematuria are caused when the tumor spreads to adjacent organs or most of renal tissues are invaded.
Treatment of renal cancer can be largely classified into surgery, chemotherapy, radiation therapy, and immunotherapy. In recent years, doctors usually remove kidney tissues with tumor parts by surgery and then carry out subsequent treatment by matching with chemotherapy or immunotherapy, but the treatment effect is not ideal, the side effect is also great, and the problems of chemotherapy failure, drug resistance and the like are difficult to solve.
Selenium is an important trace essential element present in humans and animals. Epidemiological studies, preclinical surveys and clinical intervention trials support the notion that selenium compounds can be used for cancer treatment. Patent CN107412280B discloses a nano selenium hydrosol with gastric cancer cell proliferation inhibition activity, which consists of nano selenium and coriolus versicolor polysaccharide protein, has gastric cancer cell proliferation inhibition activity, and can be used for preparing drugs for inhibiting gastric cancer cell proliferation. However, there is no report on the use of selenium for the treatment of renal cancer.
Disclosure of Invention
The invention aims to overcome the defects of the prior art, and provides a functionalized water-soluble nano-selenium, a preparation method and application thereof, which have good water solubility and can be used for preparing medicines for treating renal cancer.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
a preparation method of functional water-soluble nano-selenium comprises the following specific steps:
(1) Extracting radix pseudostellariae serving as a raw material by using a methanol aqueous solution to obtain radix pseudostellariae extract;
(2) Culturing Antrodia camphorata strains in a fermentation medium containing sodium selenite to obtain Antrodia camphorata mycelium fermentation broth;
(3) And (3) adding sodium selenite solution into the antrodia camphorate mycelium fermentation broth obtained in the step (2), stirring and mixing uniformly, then slowly adding ascorbic acid solution, stirring uniformly, heating and reacting, slowly dripping the radix pseudostellariae extract obtained in the step (1) in the reaction process, dialyzing after the reaction is finished, and freeze-drying to obtain the functionalized water-soluble nano-selenium.
Preferably, the specific method of the step (1) comprises the following steps of: firstly, pulverizing radix pseudostellariae into radix pseudostellariae powder of 100-200 meshes, then adding 1 part of radix pseudostellariae powder into 5-7 parts of 40-50% methanol aqueous solution by volume concentration, heating and refluxing for 30-40 minutes, and centrifuging to obtain a supernatant, thus obtaining the radix pseudostellariae extract.
Preferably, the specific method of step (2) is as follows: firstly, culturing Antrodia camphorata strains through an inclined plane to obtain an inclined plane full of mycelia, inoculating the inclined plane into a seed culture medium, culturing to obtain liquid seeds, inoculating the liquid seeds into a fermentation culture medium containing sodium selenite with the mass inoculum size of 5-7%, culturing to obtain a culture, performing enzymolysis, homogenizing treatment, centrifuging, and taking the supernatant to obtain the Antrodia camphorata mycelia fermentation broth.
Further preferably, the slant culture is performed on a slant culture medium under the following culture conditions: culturing for 5-7 days at the temperature of 27-29 ℃; the slant culture medium comprises the following components in parts by weight: 20-30 parts of glucose, 10-15 parts of agar, 8-10 parts of peptone, 0.3-0.4 part of magnesium sulfate and 1000 parts of potato decoction.
Further preferably, the culture conditions in the seed medium are: culturing for 2-3 days at the temperature of between 27 and 29 ℃ and at the speed of between 150 and 200 r/min; the seed culture medium comprises the following components in parts by weight: 22-30 parts of glucose, 5-7 parts of peptone, 1-2 parts of magnesium sulfate and 1000 parts of potato decoction.
Further preferably, the culture conditions in the fermentation medium are: culturing for 2-3 days at the temperature of between 27 and 29 ℃ and at the speed of between 150 and 200 r/min; the fermentation medium comprises the following components in parts by weight: 25-35 parts of glucose, 2-3 parts of peptone, 1-2 parts of magnesium sulfate, 1-2 parts of sodium selenite, 0.5-0.7 part of dipotassium hydrogen phosphate, 0.3-0.4 part of yeast powder and 1000 parts of potato decoction.
Still more preferably, the potato juice is obtained by cutting 200 to 220 parts by weight of potato, boiling in 1000 parts of water for 30 to 40 minutes, and filtering.
Further preferably, the specific method for enzymolysis comprises the following steps of: firstly, adding 0.5-0.7 part of 2X 10 enzyme activity into 100 parts of culture 5 U/kg composite proteinase, 0.4-0.6 parts enzyme activity 1X 10 5 U/kg cellulase, 0.3-0.4 part of enzyme activity 1X 10 5 U/kg amylase, carrying out enzymolysis treatment for 2-3 hours at 50-55 ℃, and inactivating enzyme.
Further preferably, the specific method of homogenizing treatment is as follows: firstly, treating for 8 to 10 minutes by a colloid mill at 1000 to 1200r/min, and then homogenizing for 2 to 3 minutes under the condition of 45 to 55 MPa.
Preferably, the specific method of the step (3) is as follows: firstly, adding 150-160 parts of 0.02-0.03 mol/L sodium selenite solution into 100 parts of antrodia camphorate mycelium fermentation broth, stirring and uniformly mixing, then slowly adding 150-160 parts of 0.04-0.05 mol/L ascorbic acid solution, stirring uniformly, heating to 50-55 ℃, keeping the temperature and stirring for 70-90 minutes, then uniformly dripping 50-60 parts of radix pseudostellariae extract solution while stirring for 30-40 minutes, keeping the temperature and stirring for 50-70 minutes after the dripping is finished, ending the reaction, dialyzing and freeze-drying to obtain the functional water-soluble nano selenium.
Preferably, in the step (3), dialysis is performed by using a dialysis bag with a molecular weight cut-off of 3000-4000 Da, the dialysis temperature is 4 ℃, and the dialysis time is 56-70 hours.
The functional water-soluble nano selenium is prepared by the preparation method.
The application of the functionalized water-soluble nano-selenium in preparing a medicament for treating renal cancer.
The invention has the beneficial effects that:
the invention firstly uses radix pseudostellariae as a raw material, and extracts the radix pseudostellariae by methanol aqueous solution to obtain radix pseudostellariae extract; culturing Antrodia camphorata strains in a fermentation medium containing sodium selenite to obtain Antrodia camphorata mycelium fermentation broth; then adding sodium selenite solution into Antrodia camphorate mycelium fermentation broth, stirring and mixing uniformly, then slowly adding ascorbic acid solution, stirring uniformly, heating and reacting, slowly dripping radix Pseudostellariae extract in the reaction process, dialyzing after the reaction is finished, and freeze-drying to obtain nano selenium. The nano selenium has good water solubility, and can be used for preparing medicines for treating renal cancer after functional modification.
The preparation method is simple, and nano selenium is generated by the reaction of sodium selenite and ascorbic acid in the environment of Antrodia camphorate mycelium fermentation broth. The Antrodia camphorate mycelium fermentation liquid is obtained by culturing Antrodia camphorate strains in a fermentation medium containing sodium selenite, so that selenium in the fermentation medium is enriched and converted into organic selenium in the growth process of the Antrodia camphorate mycelium. Selenium exists in the Antrodia camphorate mycelium fermentation liquid, which is helpful for controlling the reaction rhythm of sodium selenite and ascorbic acid, controlling the speed, the size and the shape of generating nano-selenium and avoiding the agglomeration of nano-selenium in the reaction process.
Polysaccharide substances are contained in the Antrodia camphorate mycelium fermentation broth, so that the polysaccharide substances can be subjected to surface modification along with the generation of nano selenium, and the water solubility of the nano selenium is improved. And the radix pseudostellariae extracting solution is slowly dripped in the reaction process, and the cyclic peptide contained in the radix pseudostellariae extracting solution can further modify the nano-selenium, so that the water solubility of the nano-selenium is further improved.
In addition, polysaccharide in Antrodia camphorate mycelium fermentation broth and cyclic peptide in radix pseudostellariae extract have synergistic effect with nano-selenium, and have obvious inhibition effect on renal cancer cells, so the nano-selenium obtained by the invention has great application possibility in preparing medicines for treating renal cancer.
Detailed Description
The present invention will be further illustrated by the following examples, which are given by way of illustration only and are not intended to be limiting.
The Antrodia camphorata strain, ATCC200183, is purchased from American type culture Collection; a498 human kidney cancer cells, purchased from enzyme-linked (Shanghai) biological reagent technologies, inc.
Example 1:
a preparation method of functional water-soluble nano-selenium comprises the following specific steps:
(1) Pulverizing radix Pseudostellariae into 100 mesh radix Pseudostellariae powder, adding 1g radix Pseudostellariae powder into 7g 40% methanol water solution, heating and refluxing for 40 min, centrifuging to obtain supernatant, and obtaining radix Pseudostellariae extractive solution;
(2) Culturing Antrodia camphorata strain ATCC200183 by inclined plane to obtain inclined plane full of mycelium, inoculating the inclined plane into a seed culture medium, culturing to obtain liquid seeds, inoculating the liquid seeds into a fermentation culture medium containing sodium selenite with the mass inoculum size of 5%, culturing to obtain a culture, performing enzymolysis, homogenizing treatment, centrifuging, and taking the supernatant to obtain an Antrodia camphorata mycelium fermentation broth;
(3) Adding 160g of 0.02mol/L sodium selenite solution into 100g of Antrodia camphorate mycelium fermentation broth, stirring and mixing uniformly, then slowly adding 160g of 0.04mol/L ascorbic acid solution, stirring uniformly, heating to 55 ℃, preserving heat and stirring for 70 minutes, then dropwise adding 50g of radix pseudostellariae extract at a constant speed for 40 minutes while stirring, continuing to preserving heat and stirring for 70 minutes after dropwise adding, ending the reaction, dialyzing, and freeze-drying to obtain the functional water-soluble nano-selenium.
In the step (2), slant culture is realized on a slant culture medium, and the culture conditions are as follows: culturing at 27 ℃ for 7 days; the slant culture medium comprises: glucose 20g, agar 15g, peptone 8g, magnesium sulfate 0.4g and potato juice 1000g.
The culture conditions in the seed medium were: culturing at 29 deg.C for 2 days at 150 r/min; the seed culture medium comprises: 30g of glucose, 5g of peptone, 2g of magnesium sulfate and 1000g of potato decoction.
The culture conditions in the fermentation medium were: culturing at 29 deg.C for 2 days at 150 r/min; the fermentation medium comprises: 35g of glucose, 2g of peptone, 2g of magnesium sulfate, 1g of sodium selenite, 0.7g of dipotassium hydrogen phosphate, 0.3g of yeast powder and 1000g of potato decoction.
The potato juice is obtained by cutting 220g of potato, putting into 1000g of water, boiling for 30 minutes, and filtering.
The enzymolysis method comprises the following steps: to 100g of the culture, 0.7g of an enzyme activity 2X 10 was added 5 U/kg complex protease, 0.4g enzyme activity 1×10 5 U/kg cellulase, 0.4g enzyme activity 1X 10 5 U/kg amylase, performing enzymolysis at 50 ℃ for 3 hours, and inactivating the enzyme.
The specific method for homogenizing treatment comprises the following steps: the treatment is carried out for 10 minutes by a colloid mill at 1000r/min, and then the homogenization treatment is carried out for 3 minutes under the condition of 45 MPa.
In the step (3), dialysis is carried out by using a dialysis bag with a molecular weight cut-off of 3000Da, the dialysis temperature is 4 ℃, and the dialysis time is 70 hours.
Example 2:
a preparation method of functional water-soluble nano-selenium comprises the following specific steps:
(1) Pulverizing radix Pseudostellariae into 200 mesh radix Pseudostellariae powder, adding 1g radix Pseudostellariae powder into 5g 50% methanol water solution, heating and refluxing for 30 min, centrifuging to obtain supernatant, and collecting radix Pseudostellariae extractive solution;
(2) Culturing Antrodia camphorata strain ATCC200183 by inclined plane to obtain inclined plane full of mycelium, inoculating the inclined plane into a seed culture medium, culturing to obtain liquid seeds, inoculating the liquid seeds into a fermentation culture medium containing sodium selenite with the mass inoculum size of 7%, culturing to obtain a culture, performing enzymolysis, homogenizing treatment, centrifuging, and taking the supernatant to obtain an Antrodia camphorata mycelium fermentation broth;
(3) Adding 150g of 0.03mol/L sodium selenite solution into 100g of Antrodia camphorate mycelium fermentation broth, stirring and mixing uniformly, then slowly adding 150g of 0.05mol/L ascorbic acid solution, stirring uniformly, heating to 50 ℃, preserving heat and stirring for 90 minutes, then dropwise adding 60g of radix pseudostellariae extract at a constant speed for 30 minutes while stirring, continuing to preserving heat and stirring for 50 minutes after dropwise adding, ending the reaction, dialyzing, and freeze-drying to obtain the functional water-soluble nano-selenium.
In the step (2), slant culture is realized on a slant culture medium, and the culture conditions are as follows: culturing at 29 ℃ for 5 days; the slant culture medium comprises: 30g of glucose, 10g of agar, 10g of peptone, 0.3g of magnesium sulfate and 1000g of potato decoction.
The culture conditions in the seed medium were: culturing at a temperature of 200r/min and a temperature of 27 ℃ for 3 days; the seed culture medium comprises: 22g of glucose, 7g of peptone, 1g of magnesium sulfate and 1000g of potato decoction.
The culture conditions in the fermentation medium were: culturing at a temperature of 200r/min and a temperature of 27 ℃ for 3 days; the fermentation medium comprises: 25g of glucose, 3g of peptone, 1g of magnesium sulfate, 2g of sodium selenite, 0.5g of dipotassium hydrogen phosphate, 0.4g of yeast powder and 1000g of potato decoction.
The potato juice is obtained by cutting 200g of potato, putting into 1000g of water, boiling for 40 minutes, and filtering.
The enzymolysis method comprises the following steps: to 100g of the culture, 0.5g of an enzyme activity 2X 10 was added 5 U/kg complex protease, 0.6g enzyme activity 1×10 5 U/kg cellulase, 0.3g enzyme activity 1X 10 5 U/kg amylase, and performing enzymolysis at 55 ℃ for 2 hours, and inactivating the enzyme.
The specific method for homogenizing treatment comprises the following steps: the treatment is carried out for 8 minutes by a colloid mill at 1200r/min, and then the homogenization treatment is carried out for 2 minutes under the condition of 55 MPa.
In the step (3), dialysis is carried out by using a dialysis bag with the molecular weight cut-off of 4000Da, the dialysis temperature is 4 ℃, and the dialysis time is 56 hours.
Example 3:
a preparation method of functional water-soluble nano-selenium comprises the following specific steps:
(1) Pulverizing radix pseudostellariae into 200-mesh radix pseudostellariae powder, adding 1g of radix pseudostellariae powder into 6g of 45% methanol aqueous solution with volume concentration, heating and refluxing for 30-40 minutes, and centrifuging to obtain a supernatant to obtain the radix pseudostellariae extract;
(2) Culturing Antrodia camphorata strain ATCC200183 by inclined plane to obtain inclined plane full of mycelium, inoculating the inclined plane into a seed culture medium, culturing to obtain liquid seeds, inoculating the liquid seeds into a fermentation culture medium containing sodium selenite with the mass inoculum size of 6%, culturing to obtain a culture, performing enzymolysis, homogenizing treatment, centrifuging, and taking the supernatant to obtain an Antrodia camphorata mycelium fermentation broth;
(3) Adding 155g of 0.025mol/L sodium selenite solution into 100g of Antrodia camphorate mycelium fermentation broth, stirring and uniformly mixing, then slowly adding 155g of 0.045mol/L ascorbic acid solution, stirring uniformly, heating to 52 ℃, preserving heat and stirring for 80 minutes, then dropwise adding 55g of radix pseudostellariae extract at constant speed for 35 minutes while stirring, continuing to preserving heat and stirring for 60 minutes after dropwise adding, ending the reaction, dialyzing, and freeze-drying to obtain the functional water-soluble nano-selenium.
In the step (2), slant culture is realized on a slant culture medium, and the culture conditions are as follows: culturing at 28 ℃ for 6 days; the slant culture medium comprises: 25g of glucose, 12g of agar, 9g of peptone, 0.35g of magnesium sulfate and 1000g of potato decoction.
The culture conditions in the seed medium were: culturing at 28deg.C for 3 days at 150 r/min; the seed culture medium comprises: 28g of glucose, 6g of peptone, 1.5g of magnesium sulfate and 1000g of potato decoction.
The culture conditions in the fermentation medium were: culturing at 28deg.C for 3 days at 200 r/min; the fermentation medium comprises: 30g of glucose, 2.5g of peptone, 1.5g of magnesium sulfate, 1.5g of sodium selenite, 0.6g of dipotassium hydrogen phosphate, 0.35g of yeast powder and 1000g of potato decoction.
The potato juice is obtained by cutting 210g of potato, putting into 1000g of water, boiling for 35 minutes, and filtering.
The enzymolysis method comprises the following steps: to 100g of the culture, 0.6g of an enzyme activity 2X 10 was added 5 U/kg complex protease, 0.5g enzyme activity 1×10 5 U/kg cellulase, 0.35g enzyme activity 1X 10 5 U/kg amylase, performing enzymolysis at 52 ℃ for 2.5 hours, and inactivating enzyme.
The specific method for homogenizing treatment comprises the following steps: the treatment is carried out for 9 minutes by a colloid mill at 1100r/min, and then the homogenization treatment is carried out for 2 minutes under 50 MPa.
In the step (3), dialysis is carried out by adopting a dialysis bag with the molecular weight cut-off of 3500Da, the dialysis temperature is 4 ℃, and the dialysis time is 65 hours.
Comparative example 1
A preparation method of nano-selenium comprises the following specific steps:
(1) Culturing Antrodia camphorata strain ATCC200183 by inclined plane to obtain inclined plane full of mycelium, inoculating the inclined plane into a seed culture medium, culturing to obtain liquid seeds, inoculating the liquid seeds into a fermentation culture medium containing sodium selenite with the mass inoculum size of 5%, culturing to obtain a culture, performing enzymolysis, homogenizing treatment, centrifuging, and taking the supernatant to obtain an Antrodia camphorata mycelium fermentation broth;
(2) Adding 160g of 0.02mol/L sodium selenite solution into 100g of Antrodia camphorate mycelium fermentation broth, stirring and mixing uniformly, then slowly adding 160g of 0.04mol/L ascorbic acid solution, stirring uniformly, heating to 55 ℃, preserving heat and stirring for 180 minutes, dialyzing, and freeze-drying to obtain the nano-selenium.
Wherein, in the step (1), the slant culture is realized on a slant culture medium, and the culture conditions are as follows: culturing at 27 ℃ for 7 days; the slant culture medium comprises: glucose 20g, agar 15g, peptone 8g, magnesium sulfate 0.4g and potato juice 1000g.
The culture conditions in the seed medium were: culturing at 29 deg.C for 2 days at 150 r/min; the seed culture medium comprises: 30g of glucose, 5g of peptone, 2g of magnesium sulfate and 1000g of potato decoction.
The culture conditions in the fermentation medium were: culturing at 29 deg.C for 2 days at 150 r/min; the fermentation medium comprises: 35g of glucose, 2g of peptone, 2g of magnesium sulfate, 1g of sodium selenite, 0.7g of dipotassium hydrogen phosphate, 0.3g of yeast powder and 1000g of potato decoction.
The potato juice is obtained by cutting 220g of potato, putting into 1000g of water, boiling for 30 minutes, and filtering.
The enzymolysis method comprises the following steps: to 100g of the culture, 0.7g of an enzyme activity 2X 10 was added 5 U/kg complex protease, 0.4g enzyme activity 1×10 5 U/kg cellulase, 0.4g enzyme activity 1X 10 5 U/kg amylase, performing enzymolysis at 50 ℃ for 3 hours, and inactivating the enzyme.
The specific method for homogenizing treatment comprises the following steps: the treatment is carried out for 10 minutes by a colloid mill at 1000r/min, and then the homogenization treatment is carried out for 3 minutes under the condition of 45 MPa.
In the step (2), dialysis is carried out by using a dialysis bag with a molecular weight cut-off of 3000Da, the dialysis temperature is 4 ℃, and the dialysis time is 70 hours.
Comparative example 2
A preparation method of nano-selenium comprises the following specific steps:
(1) Pulverizing radix Pseudostellariae into 100 mesh radix Pseudostellariae powder, adding 1g radix Pseudostellariae powder into 7g 40% methanol water solution, heating and refluxing for 40 min, centrifuging to obtain supernatant, and obtaining radix Pseudostellariae extractive solution;
(2) Adding 160g of 0.02mol/L sodium selenite solution into 100g of radix pseudostellariae extract, stirring and mixing uniformly, then slowly adding 160g of 0.04mol/L ascorbic acid solution, stirring uniformly, heating to 55 ℃, preserving heat and stirring for 180 minutes, dialyzing, and freeze-drying to obtain the nano-selenium.
In the step (2), a dialysis bag with a molecular weight cut-off of 3000Da is adopted for dialysis, the dialysis temperature is 4 ℃, and the dialysis time is 70 hours.
Comparative example 3
A preparation method of nano-selenium comprises the following specific steps:
(1) Pulverizing radix Pseudostellariae into 100 mesh radix Pseudostellariae powder, adding 1g radix Pseudostellariae powder into 7g 40% methanol water solution, heating and refluxing for 40 min, centrifuging to obtain supernatant, and obtaining radix Pseudostellariae extractive solution;
(2) Culturing Antrodia camphorata strain ATCC200183 by inclined plane to obtain inclined plane full of mycelium, inoculating the inclined plane into a seed culture medium, culturing to obtain liquid seeds, inoculating the liquid seeds into a fermentation culture medium containing sodium selenite with the mass inoculum size of 5%, culturing to obtain a culture, performing enzymolysis, homogenizing treatment, centrifuging, and taking the supernatant to obtain an Antrodia camphorata mycelium fermentation broth;
(3) Stirring and mixing 100g of Antrodia camphorate mycelium fermentation liquor and 50g of radix pseudostellariae extract evenly, adding 160g of 0.02mol/L sodium selenite solution, stirring and mixing evenly, slowly adding 160g of 0.04mol/L ascorbic acid solution, stirring evenly, heating to 55 ℃, preserving heat and stirring for 180 minutes, dialyzing, and freeze-drying to obtain the nano-selenium.
In the step (2), slant culture is realized on a slant culture medium, and the culture conditions are as follows: culturing at 27 ℃ for 7 days; the slant culture medium comprises: glucose 20g, agar 15g, peptone 8g, magnesium sulfate 0.4g and potato juice 1000g.
The culture conditions in the seed medium were: culturing at 29 deg.C for 2 days at 150 r/min; the seed culture medium comprises: 30g of glucose, 5g of peptone, 2g of magnesium sulfate and 1000g of potato decoction.
The culture conditions in the fermentation medium were: culturing at 29 deg.C for 2 days at 150 r/min; the fermentation medium comprises: 35g of glucose, 2g of peptone, 2g of magnesium sulfate, 1g of sodium selenite, 0.7g of dipotassium hydrogen phosphate, 0.3g of yeast powder and 1000g of potato decoction.
The potato juice is obtained by cutting 220g of potato, putting into 1000g of water, boiling for 30 minutes, and filtering.
The enzymolysis method comprises the following steps: to 100g of the culture, 0.7g of an enzyme activity 2X 10 was added 5 U/kg complex protease, 0.4g enzyme activity 1×10 5 U/kg cellulase, 0.4g enzyme activity 1X 10 5 U/kg amylase, performing enzymolysis at 50 ℃ for 3 hours, and inactivating the enzyme.
The specific method for homogenizing treatment comprises the following steps: the treatment is carried out for 10 minutes by a colloid mill at 1000r/min, and then the homogenization treatment is carried out for 3 minutes under the condition of 45 MPa.
In the step (3), dialysis is carried out by using a dialysis bag with a molecular weight cut-off of 3000Da, the dialysis temperature is 4 ℃, and the dialysis time is 70 hours.
Test examples
1. Water solubility study:
the nano-selenium obtained in examples 1 to 3 and comparative examples 1 to 3 were each added to 100mL of 8℃water at 1g, and the dissolution was visually examined, and the results are shown in Table 1.
TABLE 1 Water solubility investigation results
| Dissolution in water at 8 DEG C | |
| Example 1 | Completely dissolve, clear and transparent |
| Example 2 | Completely dissolve, clear and transparent |
| Example 3 | Completely dissolve, clear and transparent |
| Comparative example 1 | Poor solubility, large amount of floccules, and precipitate at the bottom |
| Comparative example 2 | Poor solubility, large amount of floccules, and precipitate at the bottom |
| Comparative example 3 | Substantially dissolved with a small amount of floc |
As is clear from Table 1, the nanoselenium obtained in examples 1 to 3 has good water solubility. Comparative example 1 omits the radix pseudostellariae extract, comparative example 2 omits the antrodia camphorate mycelium, comparative example 3 changes the feeding time of the radix pseudostellariae extract, the water solubility of the obtained nano selenium is obviously poor, and the proper coordination modification of polysaccharide substances, cyclic peptides and the like is demonstrated to synergistically improve the water solubility of the nano selenium.
2. MTT method for evaluating growth inhibition effect of nano selenium on human kidney cancer cells
Cells in log phase of growth: a498 human renal carcinoma cells at 1.5x10 4 Concentration species were in 96-well plates. Placing at 37deg.C with 5% CO by volume 2 After culturing in an incubator for 24 hours, the original culture medium is sucked. The test was divided into a blank control group and a drug treatment group. The blank group replaced 1640 medium containing 10% fetal bovine serum by volume; the drug treatment groups were replaced with examples 1 to 3 or with a drug containing concentration of 1. Mu.MCulture mediums of nano-selenium obtained in comparative examples 1 to 3. After 48 hours of cultivation, MTT with a concentration of 5mg/mL was added and the mixture was kept under CO 2 The incubator was incubated for 4 hours, 100. Mu.L of the supernatant was then aspirated along the upper portion of the broth, 100. Mu.L of LDMSO was added, and the mixture was left in the dark for 10 minutes, and the absorbance (wavelength: 560 nm) was measured by using an enzyme-labeled instrument, and the cell viability was calculated from the absorbance, with 6 replicate wells per treatment. Cell viability (%) = Δod drug treatment/Δod blank x 100. The results are shown in Table 2.
TABLE 2 comparison of human renal cancer cell survival rates
| Cell viability (%) | |
| Example 1 | 13.12 |
| Example 2 | 13.05 |
| Example 3 | 12.93 |
| Comparative example 1 | 22.87 |
| Comparative example 2 | 25.69 |
| Comparative example 3 | 18.73 |
As can be seen from table 2, the nano-selenium obtained in examples 1 to 3 has a remarkable inhibitory effect on the growth of human kidney cancer cells, and the cell viability is low.
Comparative example 1 omits the radix pseudostellariae extract, comparative example 2 omits the antrodia camphorate mycelium, comparative example 3 changes the feeding time of the radix pseudostellariae extract, the inhibition effect of the nano selenium obtained in comparative examples 1-3 on human kidney cancer cells is poor, and proper coordination modification of polysaccharide substances, cyclic peptides and the like is demonstrated to jointly promote the inhibition of human kidney cancer cells.
While the foregoing describes the embodiments of the present invention, it is not intended to limit the scope of the present invention, and various modifications or variations may be made by those skilled in the art without the need for inventive effort on the basis of the technical solutions of the present invention.
Claims (6)
1. The preparation method of the functional water-soluble nano-selenium is characterized by comprising the following specific steps:
(1) Extracting radix pseudostellariae serving as a raw material by using a methanol aqueous solution to obtain radix pseudostellariae extract;
(2) Culturing Antrodia camphorata strains in a fermentation medium containing sodium selenite to obtain Antrodia camphorata mycelium fermentation broth;
(3) Adding 150-160 parts of 0.02-0.03 mol/L sodium selenite solution into 100 parts of antrodia camphorate mycelium fermentation broth, stirring and mixing uniformly, then slowly adding 150-160 parts of 0.04-0.05 mol/L ascorbic acid solution, stirring uniformly, heating to 50-55 ℃, keeping the temperature and stirring for 70-90 minutes, then controlling the stirring time to take 30-40 minutes while uniformly dripping 50-60 parts of radix pseudostellariae extract, keeping the temperature and stirring for 50-70 minutes after the dripping is finished, ending the reaction, dialyzing and freeze-drying to obtain the functional water-soluble nano selenium;
the specific method of the step (2) is as follows: firstly, culturing Antrodia camphorata strains through an inclined plane to obtain an inclined plane full of mycelia, inoculating the inclined plane into a seed culture medium, culturing to obtain liquid seeds, inoculating the liquid seeds into a fermentation culture medium containing sodium selenite with the mass inoculum size of 5-7%, culturing to obtain a culture, performing enzymolysis, homogenizing treatment, centrifuging, and taking the supernatant to obtain an Antrodia camphorata mycelia fermentation broth;
the enzymolysis method comprises the following steps of: firstly, adding 0.5-0.7 part of 2X 10 enzyme activity into 100 parts of culture 5 U/kg composite proteinase, 0.4-0.6 parts enzyme activity 1X 10 5 U/kg cellulase, 0.3-0.4 part of enzyme activity 1X 10 5 U/kg amylase, carrying out enzymolysis treatment for 2-3 hours at 50-55 ℃, and inactivating enzyme.
2. The preparation method according to claim 1, wherein the specific method of the step (1) is as follows in parts by weight: firstly, pulverizing radix pseudostellariae into radix pseudostellariae powder of 100-200 meshes, then adding 1 part of radix pseudostellariae powder into 5-7 parts of 40-50% methanol aqueous solution by volume concentration, heating and refluxing for 30-40 minutes, and centrifuging to obtain a supernatant, thus obtaining the radix pseudostellariae extract.
3. The preparation method according to claim 1, wherein the specific method of the homogenization treatment is: firstly, treating for 8 to 10 minutes by a colloid mill at 1000 to 1200r/min, and then homogenizing for 2 to 3 minutes under the condition of 45 to 55 MPa.
4. The method according to claim 1, wherein in step (3), dialysis is performed using a dialysis bag having a molecular weight cut-off of 3000 to 4000Da, the dialysis temperature being 4 ℃ and the dialysis time being 56 to 70 hours.
5. A functionalized water-soluble nano-selenium obtained by the method of any of claims 1-4.
6. The use of a functionalized water-soluble nano-selenium according to claim 5 in the preparation of a medicament for treating renal cancer.
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