CN117298177B - Natural immunomodulator and preparation method thereof - Google Patents
Natural immunomodulator and preparation method thereof Download PDFInfo
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- CN117298177B CN117298177B CN202311595881.1A CN202311595881A CN117298177B CN 117298177 B CN117298177 B CN 117298177B CN 202311595881 A CN202311595881 A CN 202311595881A CN 117298177 B CN117298177 B CN 117298177B
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- Prior art keywords
- valienamine
- fatty acid
- hexoside
- preparation
- compound
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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/37—Celastraceae (Staff-tree or Bittersweet family), e.g. tripterygium or spindletree
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- 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
- A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
- A61K31/7034—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
- A61K31/7036—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin having at least one amino group directly attached to the carbocyclic ring, e.g. streptomycin, gentamycin, amikacin, validamycin, fortimicins
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/66—Microorganisms or materials therefrom
- A61K35/74—Bacteria
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/66—Microorganisms or materials therefrom
- A61K35/74—Bacteria
- A61K35/741—Probiotics
- A61K35/742—Spore-forming bacteria, e.g. Bacillus coagulans, Bacillus subtilis, clostridium or Lactobacillus sporogenes
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- A—HUMAN NECESSITIES
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Abstract
The invention relates to the technical field of natural medicines, in particular to a natural immunomodulator and a preparation method thereof, wherein the natural immunomodulator comprises a microbial fermentation preparation, a fatty acid-valienamine-hexoside compound or pharmaceutically acceptable salt, solvate or hydrate thereof. The invention also provides a preparation method of the natural immunomodulator, and a biological activity experiment shows that the natural immunomodulator has an immunoregulation effect, can inhibit the expression of TNF-alpha and IL-17, can be used for preventing and/or treating immune related diseases, and has a good attenuation effect.
Description
Technical Field
The invention relates to the technical field of natural medicines, in particular to a natural immunomodulator and a preparation method thereof.
Background
The balance of the immune system is critical to physical health and is susceptible to immune disease when it is broken. Tumors and infections are associated with immune hypofunction in the body; the immune response may also cause organ transplant immune rejection, allergy and autoimmune diseases. Immunomodulators can treat immune disorders caused by immune dysfunction by modulating the immune response of the body. For diseases such as tumor and infection related to hypoimmunity, the intervention can be performed by adopting regulator for enhancing organism immunity. For diseases related to the excessive immune reaction of the organism, such as organ transplantation immune rejection, allergy, autoimmune diseases and the like, immunosuppressants can be used for improving disease symptoms and prognosis. The tripterygium wilfordii is a perennial vine of tripterygium in the family of the dulidae, has the effects of clearing heat and detoxicating, detumescence, removing food retention, disinsection, stopping bleeding and the like, has remarkable immunosuppressive activity, and is used for treating autoimmune diseases such as rheumatoid arthritis, chronic nephritis, lupus erythematosus, psoriasis and the like in the beginning of the 50 th century. The active ingredients of radix Tripterygii Wilfordii include triptolide, tripterine, etc. Although having remarkable pharmacological activity, the composition has strong killing effect on normal cells, poor water solubility and narrow therapeutic window, and greatly limits clinical application. Therefore, the pharmacological activity of the tripterygium wilfordii is improved or maintained, the toxicity of the tripterygium wilfordii to normal cells is reduced, the therapeutic index of the tripterygium wilfordii is improved, and the development of a novel high-efficiency low-toxicity immunomodulator is a technical problem to be solved.
Disclosure of Invention
Aiming at the technical problems, the invention provides a natural immunomodulator and a preparation method thereof, wherein microorganisms are adopted to ferment traditional Chinese medicines with immunoregulation effects such as tripterygium wilfordii, and the natural immunomodulator has the effects of synergism, detoxification and the like; the trichoderma harzianum can decompose cellulose into glucose for use by aeromonas hydrophila and bacillus mycoides, and is converted into amino sugar with an immunoregulation function, the amino sugar is combined with valienamine by glycosidic bond, and then a fatty acid chain is introduced, so that the obtained fatty acid-valienamine-hexoside has stronger biological affinity, and the immunoregulation effect can be improved.
The invention is realized by the following technical scheme:
a natural immunomodulator comprises microbial fermentation preparation and fatty acid-valienamine-hexoside.
Further, the preparation method of the microbial fermentation preparation comprises the following steps:
(1) Mixing and cleaning radix Tripterygii Wilfordii, radix Paeoniae alba and Corni fructus at a mass ratio of 5:1:1, drying and pulverizing at 55deg.C, sieving with 80 mesh sieve, mixing with water, sterilizing at 121deg.C for 30min, and adding K 2 HPO 4 、KH 2 PO 4 NaCl and MgSO 4 Obtaining a fermentation medium;
(2) Inoculating Trichoderma harzianum, aeromonas hydrophila and bacillus mycoides into the fermentation culture medium obtained in the step (1), wherein the inoculum size is 5%, the fermentation temperature is 26-30 ℃, and the shaking table is cultivated for 96 hours at the rotation speed of a shaking table of 150-200r/min to obtain fermentation liquor;
(3) Centrifuging the fermentation liquor obtained in the step (2) for 10min at 3000r/min, carrying out solid-liquid separation to obtain a precipitate and a supernatant I, placing the precipitate in a sterile tube, adding anhydrous methanol, carrying out ultrasonic extraction at 30 ℃ and 500W for 3-4h, cooling to room temperature, centrifuging for 10min at 12000r/min, separating to obtain a supernatant II, repeatedly extracting for 3 times, mixing the supernatant II each time, filtering the supernatant with a microporous membrane of 0.22 mu m to obtain a filtrate, freeze-drying the filtrate, and grinding the filtrate into powder to obtain the microbial fermentation preparation.
Further, the K is obtained in the step (1) 2 HPO 4 、KH 2 PO 4 NaCl and MgSO 4 The addition amounts of (C) were 0.7 g/L, 0.3 g/L, 0.5g/L and 0.5g/L, respectively.
Further, the trichoderma harzianum preservation address in the step (2) is China general microbiological culture collection center, and the preservation number is CGMCC 5.1213; the preservation address of aeromonas hydrophila is China general microbiological culture collection center, and the preservation number is CGMCC 1.2017; the preservation address of the bacillus mycoides is China general microbiological culture collection center, and the preservation number is CGMCC 1.10168; the strains are all disclosed strains.
Further, the mass-to-volume ratio of the precipitate to the anhydrous methanol in the step (3) is 2g to 15mL.
Further, the fatty acid-valienamine-hexoside includes a fatty acid-valienamine-hexoside compound or a pharmaceutically acceptable salt, solvate or hydrate thereof.
Further, the structural formula of the fatty acid-valienamine-hexoside compound is shown as formula 1:
formula 1.
Further, the R1 group in formula 1 is CH 3 (CH 2 ) 4 CH=CHCH 2 CH=CH(CH 2 ) 7 —、CH 3 (CH 2 ) 4 CH=CHCH 2 CH=CHCH 2 CH=CH(CH 2 ) 7 -and CH 3 (CH 2 ) 4 (CH=CH-CH 2 ) 4 (CH 2 ) 2 -at least one of them.
Further, the preparation method of the fatty acid-valienamine-hexoside compound comprises the following steps:
I. mixing valienamine and fatty acid in a molar ratio of 1:1, adding deionized water, adjusting pH to 7.5-8 with sodium bicarbonate, maintaining the temperature at 40-50deg.C, and stirring for 30min to obtain a reactant I;
adding the supernatant I and phytic acid obtained in the step (3) into the reactant I obtained in the step I, regulating the pH to 10-11 by using 1mol/L NaOH, stirring and heating to 100 ℃, heating for 4 hours, concentrating to 20% of the original volume at 60 ℃, and then spray drying to obtain powder;
III, mixing the powder obtained in the step II with ethyl acetate according to the mass ratio of 1:3, stirring for 1h, filtering, and concentrating the filtrate to dryness under the absolute pressure of 0.08MPa to obtain a crude extract;
and IV, performing silica gel column chromatography on the crude extract obtained in the step III, eluting with eluent with the volume ratio of cyclohexane to ethyl acetate of 70:30, performing medium-low pressure liquid-phase ODS column chromatography, eluting with eluent with the volume ratio of methanol to water of 90:10, concentrating to dryness under the absolute pressure of 0.08MPa, and eluting with the flow rate of 3mL/min to obtain the fatty acid-valienamine-hexoside compound.
Further, the mass ratio of valienamine to deionized water in the step I is 1:10.
Further, the volume ratio of the reactant I, the supernatant I and the phytic acid in the step II is 20:60:1.
Further, the fatty acid is at least one of linoleic acid, alpha-linolenic acid and tetraarachidonic acid.
Further, the pharmaceutically acceptable salt is a salt of the compound of formula 1 with an organic or inorganic base.
Still further, the salt formed is a sodium salt, potassium salt, calcium salt, iron salt, magnesium salt, zinc salt, aluminum salt, barium salt, or ammonium salt.
Further, the pharmaceutically acceptable solvate is a solution formed by dissolving the compound of formula 1 in a solvent.
Further, the pharmaceutically acceptable hydrate is a hydrate formed by combining the compound of formula 1 with water molecules through covalent bonds.
The invention also provides a preparation method of the natural immune regulator, which is characterized by comprising the following steps: uniformly mixing the microbial fermentation preparation and fatty acid-valienamine-hexoside in a mass ratio of 5:1 in a mixer to prepare the natural immunomodulator.
Further, the natural immunomodulator may be various clinically acceptable dosage forms including injections such as intravenous emulsion, oral solid preparations such as tablets, capsules, granules and the like.
Further, the natural immunomodulator contains an effective dose of microbial fermentation preparation and fatty acid-valienamine-hexoside.
Compared with the prior art, the invention has the following beneficial effects:
according to the invention, trichoderma harzianum, aeromonas hydrophila and bacillus mycoides are adopted to ferment traditional Chinese medicines with immunosuppressive effects such as tripterygium wilfordii, after the action of microorganisms, substances such as cellulose, lignin and the like in the cell walls of the traditional Chinese medicines are degraded, and active ingredients are released; the active ingredients of the traditional Chinese medicine are hydrolyzed into micromolecular substances, which is favorable for the absorption of organisms and enhances the drug effect. Meanwhile, after microbial fermentation, the toxicity of the traditional Chinese medicine is reduced, and the cytotoxicity of active ingredients such as triptolide and tripterine can be reduced, so that the toxic and side effects are reduced. And meanwhile, the components such as cellulose and protein in the traditional Chinese medicine can be used by microorganisms to promote the growth and propagation of the microorganisms, the traditional Chinese medicine and the microorganisms act synergistically and complement each other, the traditional Chinese medicine is fermented through the growth metabolism and bioconversion of the microorganisms, the efficacy can be improved, the toxicity and toxic and side effects of the traditional Chinese medicine are reduced, and the fermented product shows the efficacy higher than the additive efficacy of the drug property matrixes. In addition, trichoderma harzianum can produce cellulase, decompose cellulose into glucose for the utilization of aeromonas hydrophila and bacillus mycoides, on one hand, the growth and the reproduction of aeromonas hydrophila and bacillus mycoides can be promoted, on the other hand, the aeromonas hydrophila can convert glucose into galactosamine, the bacillus mycoides can convert glucose into glucosamine, both galactosamine and glucosamine are aminosugars, the immunoregulation function is realized, the aminosugars and valienamine are combined through glycosidic bonds, the product has the effects of resisting inflammation and regulating immunity, and the fatty acid chain is introduced, so that the obtained fatty acid-valienamine-hexoside has stronger biological affinity and can improve the immunoregulation effect.
Drawings
In order to more clearly illustrate the invention or the technical solutions of the prior art, the drawings which are used in the description of the embodiments or the prior art will be briefly described, it being obvious that the drawings in the description below are only of the invention and that other drawings can be obtained from them without inventive effort for a person skilled in the art.
FIG. 1 is a nuclear magnetic resonance spectrum of a linoleic acid-valienamine-hexoside compound according to example 3 of the invention, wherein A is 1 H-NMR chart, B is 13 C-NMR chart;
FIG. 2 is a structural formula diagram of a linoleic acid-valienamine-hexoside compound according to example 3 of the present invention;
FIG. 3 is a graph showing the effect of the natural immunomodulators described in example 5 and comparative examples 1-2 of the invention on TNF- α levels in colon tissue of mice in each group;
FIG. 4 is a graph showing the effect of the natural immunomodulators described in example 5 and comparative examples 1-2 of the invention on IL-17 content in colon tissue of mice in each group;
FIG. 5 is a graph showing the effect of the natural immunomodulator according to example 5 and comparative examples 1-2 on L02 cell viability of human normal hepatocytes.
Detailed Description
The present invention will be further described in detail with reference to the following specific examples, but the present invention is not limited to the following examples, for the purpose of making the objects, technical solutions and advantages of the present invention more apparent.
Unless otherwise specified, the chemical reagents involved in the invention are purchased commercially, and the strain is a disclosed strain.
Example 1: the embodiment provides a microbial fermentation preparation, and a preparation method of the microbial fermentation preparation comprises the following steps:
(1) Mixing and cleaning radix Tripterygii Wilfordii, radix Paeoniae alba and Corni fructus at a mass ratio of 5:1:1, drying and pulverizing at 55deg.C, sieving with 80 mesh sieve, mixing with water, sterilizing at 121deg.C for 30min, and adding K 2 HPO 4 、KH 2 PO 4 NaCl and MgSO 4 The addition amounts are respectively 0.7 g/L, 0.3 g/L, 0.5g/L and 0.5g/L, so as to obtain a fermentation culture medium;
(2) Inoculating Trichoderma harzianum, aeromonas hydrophila and bacillus mycoides into the fermentation culture medium obtained in the step (1), wherein the inoculation amount is 5%, the fermentation temperature is 30 ℃, and the shaking table is used for shake cultivation for 96 hours at the rotation speed of a shaking table of 200r/min to obtain fermentation liquor;
(3) Centrifuging the fermentation liquor obtained in the step (2) at 3000r/min for 10min, carrying out solid-liquid separation to obtain a precipitate and a supernatant I, placing the precipitate in a sterile tube, adding anhydrous methanol, carrying out ultrasonic extraction for 4h at 30 ℃ and 500W, cooling to room temperature, centrifuging at 12000r/min for 10min, separating to obtain a supernatant II, repeatedly extracting for 3 times, mixing the supernatant II each time, filtering through a microporous filter membrane of 0.22 mu m to obtain a filtrate, freeze-drying the filtrate, and grinding the filtrate into powder to obtain the microbial fermentation preparation.
Example 2: the embodiment provides a microbial fermentation preparation, and a preparation method of the microbial fermentation preparation comprises the following steps:
(1) Mixing and cleaning radix Tripterygii Wilfordii, radix Paeoniae alba and Corni fructus at a mass ratio of 5:1:1, drying and pulverizing at 55deg.C, sieving with 80 mesh sieve, mixing with water, sterilizing at 121deg.C for 30min, and adding K 2 HPO 4 、KH 2 PO 4 NaCl and MgSO 4 The addition amounts are respectively 0.7 g/L, 0.3 g/L, 0.5g/L and 0.5g/L, so as to obtain a fermentation culture medium;
(2) Inoculating Trichoderma harzianum, aeromonas hydrophila and bacillus mycoides into the fermentation culture medium obtained in the step (1), wherein the inoculation amount is 5%, the fermentation temperature is 26 ℃, and the shaking table is cultivated for 96 hours at the rotation speed of a shaking table of 150r/min to obtain fermentation liquor;
(3) Centrifuging the fermentation liquor obtained in the step (2) at 3000r/min for 10min, carrying out solid-liquid separation to obtain a precipitate and a supernatant I, placing the precipitate in a sterile tube, adding anhydrous methanol, carrying out ultrasonic extraction for 3h at 30 ℃ and 500W, cooling to room temperature, centrifuging at 12000r/min for 10min, separating to obtain a supernatant II, repeatedly extracting for 3 times, mixing the supernatant II each time, filtering through a microporous filter membrane of 0.22 mu m to obtain a filtrate, freeze-drying the filtrate, and grinding the filtrate into powder to obtain the microbial fermentation preparation.
Example 3: the embodiment provides a fatty acid-valienamine-hexoside compound, and a preparation method of the fatty acid-valienamine-hexoside compound, which comprises the following steps:
I. mixing valienamine and fatty acid in a molar ratio of 1:1, adding deionized water, adjusting pH to 8 with sodium bicarbonate, maintaining the temperature at 50deg.C, and stirring for 30min to obtain a reactant I;
adding the supernatant I obtained in the step (3) in the example 1 and phytic acid into the reactant I obtained in the step I, regulating the pH to 11 by using 1mol/L NaOH, stirring and heating to 100 ℃, heating for 4 hours, concentrating to 20% of the original volume at 60 ℃, and then spray drying to obtain powder;
III, mixing the powder obtained in the step II with ethyl acetate according to a mass ratio of 1:3, stirring for 1: 1h, filtering, and concentrating filtrate to dryness under the absolute pressure of 0.08MPa to obtain a crude extract;
and IV, performing silica gel column chromatography on the crude extract obtained in the step III, eluting with eluent with the volume ratio of cyclohexane to ethyl acetate of 70:30, performing medium-low pressure liquid-phase ODS column chromatography, eluting with eluent with the volume ratio of methanol to water of 90:10, taking a part of the eluent, analyzing by a superconducting nuclear magnetic resonance spectrometer, concentrating the rest of the eluent under the absolute pressure of 0.08MPa until the rest of the eluent is dry, and obtaining the fatty acid-valienamine-hexoside compound when eluting, wherein the flow rate of the eluent is 3 mL/min.
The mass ratio of valienamine to deionized water in the step I of the embodiment is 1:10, and the fatty acid is linoleic acid; the volume ratio of the reactant I to the supernatant I to the phytic acid in the step II is 20:60:1; FIG. 1 is a nuclear magnetic resonance spectrum of a fatty acid-valienamine-hexoside compound prepared in this example, which shows that the molecular formula of the compound is C 32 H 56 N 2 O 8 The structural formula is shown in figure 2.
Example 4: the embodiment provides a fatty acid-valienamine-hexoside compound, and a preparation method of the fatty acid-valienamine-hexoside compound, which comprises the following steps:
I. mixing valienamine and fatty acid in a molar ratio of 1:1, adding deionized water, adjusting pH to 7.5 with sodium bicarbonate, maintaining the temperature at 40deg.C, and stirring for 30min to obtain a reactant I;
adding the supernatant I obtained in the step (3) in the example 1 and phytic acid into the reactant I obtained in the step I, regulating the pH to 10 by using 1mol/L NaOH, stirring and heating to 100 ℃, heating for 4 hours, concentrating to 20% of the original volume at 60 ℃, and then spray drying to obtain powder;
III, mixing the powder obtained in the step II with ethyl acetate according to a mass ratio of 1:3, stirring for 1: 1h, filtering, and concentrating filtrate to dryness under the absolute pressure of 0.08MPa to obtain a crude extract;
and IV, performing silica gel column chromatography on the crude extract obtained in the step III, eluting with eluent with the volume ratio of cyclohexane to ethyl acetate of 70:30, performing medium-low pressure liquid-phase ODS column chromatography, eluting with eluent with the volume ratio of methanol to water of 90:10, concentrating to dryness under the absolute pressure of 0.08MPa, and eluting with the flow rate of 3mL/min to obtain the fatty acid-valienamine-hexoside compound.
The mass ratio of valienamine to deionized water in the step I of the embodiment is 1:10, and the fatty acid is alpha-linolenic acid; the volume ratio of the reactant I, the supernatant I and the phytic acid in the step II is 20:60:1.
Example 5: the embodiment provides a natural immunomodulator, which comprises a biological fermentation preparation and a fatty acid-valienamine-hexoside compound, and also provides a preparation method of the natural immunomodulator, which comprises the following steps: the microbial fermentation preparation prepared in example 1 and the fatty acid-valienamine-hexoside compound prepared in example 3 are uniformly mixed in a mixer according to a mass ratio of 5:1 to prepare the natural immunomodulator.
Comparative example 1: the comparative example provides a natural immunomodulator comprising a traditional Chinese medicine preparation and a fatty acid-valienamine-hexoside compound.
The preparation method of the traditional Chinese medicine preparation comprises the following steps: mixing and cleaning radix Tripterygii Wilfordii, radix Paeoniae alba and Corni fructus at a mass ratio of 5:1:1, drying and pulverizing at 55deg.C, sieving with 80 mesh sieve to obtain Chinese medicinal powder, placing in a sterile tube, adding anhydrous methanol, extracting with 2 g/15 mL of Chinese medicinal powder at 30deg.C and 500W by ultrasound for 4 hr, cooling to room temperature, centrifuging at 12000r/min for 10min, separating to obtain supernatant II, repeatedly extracting for 3 times, mixing the supernatant II, filtering with 0.22 μm microporous membrane to obtain filtrate, lyophilizing the filtrate, and grinding into powder to obtain the final product.
The preparation method of the fatty acid-valienamine-hexoside compound is the same as that of example 3.
The comparative example also provides a preparation method of the natural immune modulator, which comprises the following steps: uniformly mixing the traditional Chinese medicine preparation and the fatty acid-valienamine-hexoside compound in a mass ratio of 5:1 in a mixer to prepare the natural immune regulator.
Comparative example 2: the present comparative example provides a natural immunomodulator comprising a fatty acid-valienamine reactant and a microbial fermentation formulation.
The preparation method of the fatty acid-valienamine reactant comprises the following steps: mixing valienamine and fatty acid in a molar ratio of 1:1, adding deionized water, adjusting pH to 7.5 with sodium bicarbonate, maintaining at 40deg.C, stirring for 30min, concentrating at 60deg.C to 20% of the original volume, and spray drying to obtain fatty acid-valienamine reactant.
The preparation method of the microbial fermentation preparation is the same as that of example 1.
The comparative example also provides a preparation method of the natural immune modulator, which comprises the following steps: the microbial fermentation preparation and the fatty acid-valienamine reactant are uniformly mixed in a mixer according to the mass ratio of 5:1, so as to prepare the natural immunomodulator.
Experimental example 1: the natural immunomodulators of example 5 and comparative examples 1-2 were each mixed with deionized water to prepare 0.1g/mL solutions. 50 SPF-class Kunming mice were selected, male, 7-9 weeks old, weighing (20+ -2) g, 40 of which were used as model groups, after 1 week of feeding with sterile distilled water, 3wt% DSS solution was used instead of sterile distilled water, 7 d was continued, the new DSS solution was changed daily, during which 10 other mice were used as blank groups to be fed with sterile distilled water daily, and then 40 mice of the model groups were divided into example 5 group, comparative example 1 group, comparative example 2 group and CK group, 10 of each group were respectively filled with the solutions prepared by the natural immunomodulators prepared in example 5, comparative example 1, comparative example 2 and an equal volume of sterile saline for a fixed time per day, the blank groups were filled with an equal volume of sterile saline at the same time, 16 days continuously, 1 time per day, and the mice of each group were free to drink water during the test. Mice begin fasted at the end of the 15d gavage and are sacrificed 2h after the end of the 16d gavage. The colon tissue of the mice was taken, the intestinal lumen was flushed clean with normal saline, and about 0.1g of the colon tissue was placed in liquid nitrogen for preservation. Colon tissues of 5 mice are randomly picked in each group, and before detection, the colon tissues are homogenized according to the mass ratio of PBS=1:9; centrifuging the prepared homogenate at 3000r/min for 10min, absorbing supernatant, detecting the indexes of mouse TNF-alpha and IL-17 by using an ELISA kit, and carrying out the specific operation steps strictly according to the specification of the kit. The results are shown in FIGS. 3-4.
From FIGS. 3-4, the levels of TNF-. Alpha.and IL-17 were significantly increased in the colon of the CK mice compared to the blank mice. The cytokines TNF-. Alpha.and IL-17 content were extremely significantly reduced in the groups of example 5 and comparative examples 1-2 compared to the CK group. * Indicates a very significant difference compared to the blank group, # indicates a very significant difference compared to the CK group.
The results in FIGS. 3-4 show that the natural immunomodulators of example 5 and comparative examples 1-2 have different degrees of down-regulation of the levels of pro-inflammatory factors TNF- α, IL-17 in colon tissue, as best seen in group 5 of examples. The content of pro-inflammatory factors TNF-alpha and IL-17 in colon tissues of the CK group mice is obviously increased, which indicates that the colon of the CK group mice has serious inflammatory reaction, and the natural immunomodulator can improve abnormal changes of various cytokines and tends to restore balance. The natural immunomodulator has the effect of inhibiting the expression of TNF-alpha and IL-17, and can be used as immunomodulator for preventing and treating immune related diseases, such as autoimmune diseases like rheumatoid arthritis.
Experimental example 2: placing human normal liver cell L02 at 37deg.C, and containing 5% CO 2 In the cell culture chamber of (2), RPMI-1640 medium containing 10% fetal bovine serum is used as a complete medium, every 60h passage is carried out, 10000 normal human liver cells L02 in logarithmic growth phase are inoculated into a 96-well plate per 100 mu L of each well, the average of the wells of the 96-well plate is divided into 4 groups, the natural immune regulator prepared in the example 5 and the comparative example 1-2 is respectively added, the concentration of each well is 2 mu g/mL, and a blank control group without the natural immune regulator is simultaneously arranged. Placing each group at 37deg.C, and containing 5% CO 2 Is cultured in a cell culture incubator for 48 hours. Cell absorbance values were determined using CCK-8 kit according to formula C (%) = [ (a) c -A 0 )/(A b -A 0 )]*100% of calculated cell viability, C is cell viability, A c For the absorbance values of the test group, A b For the absorbance value of the control group, A 0 Is the background absorbance value. The results are shown in FIG. 5. * Indicating a very significant difference compared to the blank control group, indicating a significant difference compared to the control group.
As can be seen from the results of FIG. 5, the cell viability of the example 5 group is not significantly changed compared with that of the blank control group, the cell viability of the comparative examples 1-2 group is significantly reduced compared with that of the control group, and the attenuation effect is poor, which indicates that the natural immunomodulator has lower cytotoxicity and is safer and more reliable.
Those of ordinary skill in the art will appreciate that: the discussion of any of the embodiments above is merely exemplary and is not intended to suggest that the scope of the invention (including the claims) is limited to these examples; the technical features of the above embodiments or in the different embodiments may also be combined within the idea of the invention, the steps may be implemented in any order and there are many other variations of the different aspects of the invention as described above, which are not provided in detail for the sake of brevity.
The present invention is intended to embrace all such alternatives, modifications and variances which fall within the broad scope of the appended claims. Therefore, any omission, modification, equivalent replacement, improvement, etc. of the present invention should be included in the scope of the present invention.
Claims (4)
1. A natural immunomodulator, which is characterized by comprising a microbial fermentation preparation and fatty acid-valienamine-hexoside;
the preparation method of the microbial fermentation preparation comprises the following steps:
(1) Mixing and cleaning radix Tripterygii Wilfordii, radix Paeoniae alba and Corni fructus at a mass ratio of 5:1:1, drying and pulverizing at 55deg.C, sieving with 80 mesh sieve, mixing with water, sterilizing at 121deg.C for 30min, and adding K 2 HPO 4 、KH 2 PO 4 NaCl and MgSO 4 The addition amounts are respectively 0.7 g/L, 0.3 g/L, 0.5g/L and 0.5g/L, so as to obtain a fermentation culture medium;
(2) Inoculating Trichoderma harzianum, aeromonas hydrophila and bacillus mycoides into the fermentation culture medium obtained in the step (1), wherein the inoculum size is 5%, the fermentation temperature is 26-30 ℃, and the shaking table is cultivated for 96 hours at the rotation speed of a shaking table of 150-200r/min to obtain fermentation liquor;
(3) Centrifuging the fermentation liquor obtained in the step (2) for 10min at 3000r/min, carrying out solid-liquid separation to obtain a precipitate and a supernatant I, placing the precipitate in a sterile tube, adding anhydrous methanol, carrying out ultrasonic extraction on the precipitate and the anhydrous methanol for 3-4h at the mass volume ratio of 2g to 15mL, cooling to room temperature, centrifuging for 10min at 12000r/min, separating to obtain a supernatant II, repeatedly extracting for 3 times, mixing the supernatant II each time, filtering through a microporous filter membrane of 0.22 mu m to obtain a filtrate, freeze-drying the filtrate, and grinding the filtrate into powder to obtain the microbial fermentation preparation;
the fatty acid-valienamine-hexoside comprises a fatty acid-valienamine-hexoside compound or a pharmaceutically acceptable salt, solvate or hydrate thereof;
the structural formula of the fatty acid-valienamine-hexoside compound is shown as formula 1:
formula 1;
the R1 group in formula 1 is CH 3 (CH 2 ) 4 CH=CHCH 2 CH=CH(CH 2 ) 7 —、
CH 3 (CH 2 ) 4 CH=CHCH 2 CH=CHCH 2 CH =CH(CH 2 ) 7 —、
CH 3 (CH 2 ) 4 (CH=CH-CH 2 ) 4 (CH 2 ) 2 -at least one of;
the preparation method of the fatty acid-valienamine-hexoside compound comprises the following steps:
I. mixing valienamine and fatty acid in a molar ratio of 1:1, adding deionized water, adjusting pH to 7.5-8 with sodium bicarbonate, maintaining the temperature at 40-50deg.C, and stirring for 30min to obtain a reactant I;
adding the supernatant I and phytic acid obtained in the step (3) into the reactant I obtained in the step I, regulating the pH to 10-11 by using 1mol/L NaOH, stirring and heating to 100 ℃, heating for 4 hours, concentrating to 20% of the original volume at 60 ℃, and then spray drying to obtain powder;
III, mixing the powder obtained in the step II with ethyl acetate according to a mass ratio of 1:3, stirring for 1h, filtering, and concentrating filtrate to dryness under the absolute pressure of 0.08MPa to obtain a crude extract;
and IV, performing silica gel column chromatography on the crude extract obtained in the step III, eluting with eluent with the volume ratio of cyclohexane to ethyl acetate of 70:30, performing medium-low pressure liquid-phase ODS column chromatography, eluting with eluent with the volume ratio of methanol to water of 90:10, concentrating to dryness under the absolute pressure of 0.08MPa, and eluting with the flow rate of 3mL/min to obtain the fatty acid-valienamine-hexoside compound.
2. The natural immune modulator according to claim 1, wherein in the step I, the mass ratio of valienamine to deionized water is 1:10, and the fatty acid is at least one of linoleic acid, alpha-linolenic acid and tetraarachidonic acid; the volume ratio of the reactant I, the supernatant I and the phytic acid in the step II is 20:60:1.
3. A natural immune modulator according to claim 2, wherein the pharmaceutically acceptable salt is a salt of a compound of formula 1 with an organic or inorganic base, and the salt formed is a sodium, potassium, calcium, iron, magnesium, zinc, aluminum, barium or ammonium salt; the pharmaceutically acceptable solvate is a solution formed by dissolving a compound of formula 1 in a solvent; the pharmaceutically acceptable hydrate is a hydrate formed by combining a compound of formula 1 with water molecules in a covalent bond.
4. A method of preparing a natural immunomodulatory agent according to any one of claims 1-3 comprising the steps of: uniformly mixing the microbial fermentation preparation and fatty acid-valienamine-hexoside in a mass ratio of 5:1 in a mixer to prepare the natural immunomodulator.
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