CN113264987B - Cyclic color-threo-valyl-isoleucyl-leucinyl peptide with antifungal and free radical scavenging activities and preparation method thereof - Google Patents
Cyclic color-threo-valyl-isoleucyl-leucinyl peptide with antifungal and free radical scavenging activities and preparation method thereof Download PDFInfo
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/64—Cyclic peptides containing only normal peptide links
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/10—Antimycotics
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- 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
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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
- C12N1/14—Fungi; Culture media therefor
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
Abstract
The invention relates to a cyclic color-threo-valyl-isoleucyl-leucinyl peptide with antifungal and free radical scavenging activities and a preparation method thereof, belonging to the technical field of extraction and preparation of biological products. The cyclic color-threo-valyl-isoleucyl-leupeptin is fifteen-membered cyclic pentapeptide formed by connecting tryptophan, threonine, valine, isoleucine and leucine through an amide bond, and is white powder; is a novel compound and has not been reported in the literature. The compound can be obtained by culturing frog-feces mould, and then extracting and separating. The pure product of the cyclochrome-threo-valyl-isoleucyl-leucinyl peptide is white powder, and the half-number scavenging concentration of DPPH free radical is as follows: 1.31 The diameter of the inhibition zone for the streptococci leucovorin at the concentration of 200 mug/mL is 13.74 and mm. The compound has the potential of preparing antibacterial drugs and antioxidant and anti-aging health care products.
Description
Technical Field
The invention belongs to the technical field of extraction and preparation of biological products, and particularly relates to extraction and purification of cyclophosphamide-threo-valyl-isoleucyl-leucinide (Trp-Thr-Val-Ile-Leu cyclopeptide) with antifungal and free radical scavenging activities.
Background
The cyclic color-threo-valyl-isoleucyl-leucinyl peptide is prepared from frog excrement mouldBasidiobolussp.) the biologically active substance with antifungal and free radical scavenging activity is isolated from the culture.
Frog feces mouldBasidiobolussp.) is a fungus of the genus frog-faecalis of the family frog-faecalis of the order Cordyceps; there are frog-derived and spore-forming frog-feces mold, solid spore frog-feces mold, and schizospore frog-feces mold, etc. which are common in our country. The strain can be derived from soil and amphibiansSeparating animal feces.
Antifungal Activity: there are a variety of fungi which are conditional pathogens and are susceptible to infection when people have reduced resistance, especially for the elderly and patients. At present, the aging in the world is more and more serious, meanwhile, due to factors such as environmental pollution and the like, the resistance of people is reduced, fungal infection cases are increased, and meanwhile, early antifungal agents have drug resistance, so that the development of novel antifungal active substances is very significant.
Scavenging free radical activity: free radical (free radical) refers to a group, atom or molecule containing unpaired electrons from a chemical structural point of view. The free radicals are highly chemically active. For living bodies, free radicals are intermediates of various biochemical reactions in vital activities. Under normal conditions, free radicals in the human body are in dynamic equilibrium with respect to production and scavenging. Free radicals are an effective defense system of the organism and can adversely affect the life activities of the organism if a certain level of free radicals cannot be maintained. However, the free radical is produced too much or eliminated too slowly, and it can cause various damages on the molecular level, the cellular level and the tissue organ level of the organism by attacking the living macromolecular substances and various organelles, accelerate the aging process of the organism and induce various diseases. Recent studies have shown that aging and many diseases in humans are associated with free radical damage. The substance with free radical scavenging activity can react with free radicals and reduce the free radicals into non-free radical compounds, can scavenge excessive free radicals generated in the metabolic process of the organism, and is an important active substance capable of improving the health of human bodies. The free radical scavenger has antioxidant effect in non-living system, can effectively prevent oxidation and deterioration of substances, has important effect in prolonging shelf life of articles, and is widely used in foods, medicines, daily chemicals, etc.
Disclosure of Invention
The invention aims to provide a cyclic color-threo-valyl-isoleucyl-leucinyl peptide with antifungal and free radical scavenging activities and a preparation method thereof.
In order to find novel natural and efficient substances with antifungal activity and free radical scavenging activity, the inventor finds that the cyclophosphamide-threo-valyl-isoleucyl-leupeptin extracted from the frog-like faecalis has strong anti-streptococci activity and free radical scavenging activity through carrying out activity research on more than 100 entomopathogenic fungi metabolites in China.
The structural formula of the cyclic color-threo-valyl-isoleucyl-leucinyl peptide with antifungal and free radical scavenging activities is as follows:
the cyclic color-threo-valyl-isoleucyl-leupeptin is fifteen-membered cyclic pentapeptide formed by connecting tryptophan, threonine, valine, isoleucine and leucine through an amide bond, and is white powder;
the cyclophosphamide-threo-valyl-isoleucyl-leupeptin has free radical scavenging activity and anti-streptoverticillium albuminosus activity;
half-scavenging concentration (DC) of P-diphenylpicrylphenylhydrazine radical (DPPH) 50 ) The method comprises the following steps: the diameter of the inhibition zone for Streptomyces albus at a concentration of 200. Mu.g/mL was 13.74mm, which was 1.31 mg/mL.
The preparation of the cyclic color-threo-valyl-isoleucyl-leucinyl peptide with antifungal and free radical scavenging activities is carried out as follows:
(1) Strain culture
The frog manure mould is preparedBasidiobolussp.) carrying out slant strain culture, secondary strain culture, tertiary strain culture and quaternary culture to obtain a liquid final fermentation product or a solid final fermentation product;
the frog manure mould is preparedBasidiobolussp.) is one of frog-derived faecalis, solid spore frog faecalis or schizospore faecalis.
(2) Extraction and refinement of active principles in the final fermentation
Extracting the effective components in the final fermentation product with methanol or ethanol, and purifying by reverse phase chromatography to obtain white powdery cyclophosphamide-threo-valyl-isoleucyl-leucinyl peptide.
The preparation technical scheme is further defined as follows:
the specific operation of the step (1) is as follows:
(1.1) slant culture of bacterial species
Inoculating the frog-fecal mould strain into potato agar (PDA) slant culture medium, and culturing for 4-8 d at constant temperature of 25-36 ℃ to obtain first-class strain;
(1.2) culturing of secondary strains
Inoculating the first-level strain to a liquid shake flask culture medium for culture;
adding a liquid culture medium with the volume of 30-50% of that of a triangular flask in the triangular flask with the volume of more than or equal to 100 ml, inoculating 1-3 inclined plane strains to each triangular flask, and culturing for 3-7 d in a full-temperature shaking incubator at the temperature of 25-36 ℃ and the rotating speed of 150-250 rpm to obtain a secondary strain;
the liquid shake flask culture medium consists of glucose 25.0-50.0 g/L, malt extract 10.0-30.0 g/L, peptone 2.0-6.0 g/L, yeast extract 1.0-4.0 g/L, potassium chloride (KCl) 2 ) 0.3-2.0 g/L, magnesium sulfate heptahydrate (MgSO) 4 .7H 2 O) 0.3-2.0 g/L, monopotassium phosphate (KH) 2 PO 4 ）0.3～2.0 g/L；
(1.3) three-stage Strain culture
Inoculating the second-level strain to a fermentation tank with the volume of more than or equal to 5L, wherein the liquid loading amount is 50-80% of the tank volume, the inoculation amount is 3-10%, and carrying out constant-temperature aeration culture for 2-5 days under the conditions that the aeration rate is 1:1-2 (v/v), the pressure is 0.1-0.2 MP and the temperature is 25-36 ℃ to obtain a third-level strain;
the medium in the fermenter is the liquid medium in step (1.2);
(1.4) four-stage culture
The four-stage culture is liquid culture, three-stage strains are inoculated into a fermentation tank with the volume of more than or equal to 50L, the liquid loading amount is 50-80 percent of the volume of the tank, the inoculum size is 3-10 percent, and the final fermentation product of the liquid is obtained by constant-temperature aeration culture for 5-15 days under the conditions that the aeration rate is 1:1-2 (v/v), the pressure is 0.1-0.2 MP and the temperature is 25-36 ℃; culture medium in fermentors the liquid culture medium in step (1.2).
In the step (1.4), the four-stage culture is solid culture, the three-stage liquid strain is mixed into a solid culture medium, and the inoculation amount is calculatedCulturing at 25-36 deg.c for 5-15 days to obtain solid final fermented matter in 3-10 wt% of solid material; the solid culture medium consists of glucose 25.0-50.0 g/L, malt extract 10.0-30.0 g/L, peptone 2.0-6.0 g/L, yeast extract 1.0-4.0 g/L and potassium chloride (KCl) 2 ) 0.3-2.0 g/L, magnesium sulfate heptahydrate (MgSO) 4 .7H 2 O) 0.3-2.0 g/L, monopotassium phosphate (KH) 2 PO 4 ) 0.3 to 2.0g/L, 15.0 to 30.0g/L of agar and water.
The specific operation of the step (2) is as follows:
(2.1) pretreatment of the final fermentation product
Filtering the final fermentation product of the liquid by a 0.20-1.0 mu m filter membrane or centrifuging at 2000-12000 r/min to obtain mycelium; freeze-drying mycelium, and pulverizing to obtain a pretreated substance passing through a 20-120 mesh sieve;
or, drying the final fermentation product of the solid at 30-130 ℃ until the water content is less than or equal to 20%, and then crushing the final fermentation product into a pretreated product of 20-120 meshes;
(2.2) extraction, separation and purification of the active ingredient in the pretreated matter
(2.2.1) extraction
Extracting the pretreated material with methanol or ethanol; the ratio of the feed to the liquid is 1:2-20 (W: V), the ultrasonic power is 10-100 KHz, and the extraction time is 10-200 minutes; centrifuging at a rotation speed of 2000 rpm or more, or filtering with a 0.20-1.0 μm filter membrane; concentrating the filtrate or the centrifugal supernatant to 1/2-1/10 of the original volume at the temperature of less than or equal to 100 ℃ to obtain concentrated dealcoholized liquid, and simultaneously recovering methanol or ethanol, wherein the concentrated dealcoholized liquid is used for further chromatographic refining;
(2.2.2) separation and purification
Purifying by adopting a reversed phase chromatography method, wherein the stationary phase is a bonding phase filler, the bonding phase filler is reversed phase carbon eighteen filler (RPC 18), and the particle diameter is 3-50 microns; methanol and water are used according to the proportion of 0 to 100: gradient elution is carried out according to the proportion of 100-0, and chromatographic flow with the molecular weight of 676 on a mass spectrum detector is collected; concentrating the collected matter at 100 deg.c or lower to viscous state, and drying at 170 deg.c or lower; the cyclic-threo-valo-isoleucyl-leucinide is obtained as a white powder.
The analytical study of the present invention is described as follows:
1. screening and researching to find out frog-excrement mouldBasidiobolussp.) mycelium methanol extract has stronger activity of scavenging free radicals and resisting streptococci albicans:
(1) Determination of the inhibitory Activity of methanol extract against Streptomyces albus: preparing 20% DMSO solution with the concentration of 1.0 mg/ml, taking amphotericin B as positive control, taking 20% DMSO solution as blank control, performing experiment for inhibiting Alternaria albus, and calculating to obtain the average inhibition zone diameter of 11.53 mm of the extract on Alternaria albus.
(2) Determination of scavenging free radical Activity of methanol extract: the concentration of the extract samples was 0.2, 0.4, 0.8, 1.0 and 1.6. 1.6 mg/mL methanol solution, the scavenging activity of the extract on DPPH free radical of 0.5 mmol/L was measured at 517. 517 nm, and the half-value scavenging concentration of DPPH free radical was calculated to be 1.49. 1.49 mg/mL.
Since the methanol extract has a low content of active ingredients, further extraction and purification of the active ingredients are required.
2. Further separating and purifying the methanol extract by reverse phase chromatography to obtain a pure product with the following biological activity:
(1) Determination of the inhibitory Activity of Cyclochrome-Su-Val-Isopenia-leucinum on Streptomyces albus: preparing 20% DMSO solution with extract concentration of 0.2 mg/ml, taking amphotericin B as positive control, taking 20% DMSO solution as blank control, performing test for inhibiting Alternaria albus, and calculating to obtain average inhibition zone diameter of extract on Alternaria albus of 13.74mm.
(2) Determination of scavenging free radical Activity of cyclophospha-threo-valo-isoleucyl-leucinyl peptide: the concentration of the extract samples was 0.2, 0.4, 0.8, 1.0 and 1.6. 1.6 mg/mL methanol solution, the scavenging activity of the extract on DPPH free radical of 0.5 mmol/L was measured at 517. 517 nm, and the half-value scavenging concentration of DPPH free radical was calculated to be 1.31. 1.31 mg/mL.
3. Chemical structure identification of active compounds
High scoreAnalysis by liquid chromatography-mass spectrometry shows that the purified active compound positive ion 585.3440 (M+H) + )、607.3261(M+Na + )，623.3000(M+K + ) The calculated molecular formula is C 30 H 44 N 6 O 6 。
The nmr analysis data are shown in the following table:
the active compound structure obtained by separation and purification through comprehensive liquid chromatography-mass spectrometry analysis and nuclear magnetic resonance analysis is the cyclic color-threo-valyl-isoleucyl-leucinyl peptide, and the chemical structural formula is shown.
The beneficial technical effects of the invention are shown in the following aspects:
1. the cyclophosphamide-threo-valyl-isoleucyl-leupeptin prepared by the method has the activities of inhibiting the streptococci albicans and scavenging free radicals, and opens up a new application field of the frog-faecalis. The invention discovers for the first time that the frog-faecalis has the function of metabolizing the active substances. On the basis of the invention, the related genes can be expected to be cloned further, and high-yield strains can be constructed.
2. The cyclophosphamide-threo-valyl-isoleucyl-leucinyl peptide is a novel framework, can be used as a drug lead compound for derivatization and transformation, and can be used for creating compounds with more activities and stronger activity.
3. The cyclophosphamide-threo-valyl-isoleucyl-leucinide can be used for preparing a streptococcal inhibitor and a free radical scavenger. The free radical scavenger has effects of whitening, resisting oxidation, aging, fresh keeping, sterilizing, and relieving inflammation, and the white streptococcal inhibitor prepared from cyclophosphamide-threo-valyl-isoleucyl-leucinide can be used for preventing and treating fungal infection.
4. The invention is not influenced by environment and resources due to the adoption of microbial fermentation production, is easy to realize industrialization and automation, and is not influenced by environment and natural resources.
5. The product produced by the process method has low cost, simple and convenient process, stable process, easy regulation and control and high success rate.
Detailed Description
The invention is further described below with reference to examples.
Example 1:
the preparation method of the cyclophilin-threo-valyl-isoleucyl-leucinyl peptide with antifungal and free radical scavenging activities comprises the following steps:
the frog-excrement mould used in this example 1 is frog-raw frog-excrement mould.
(1) Strain culture
(1.1) slant seed culture
Inoculating frog-derived faecal mould strain into potato agar (PDA) slant culture medium, and culturing at 25deg.C for 4d to obtain first-class strain;
(1.2) culturing of secondary strains
Inoculating the first-level strain to a liquid shake flask culture medium for culture;
liquid shake flask medium: 25.0g/L glucose, 10.0g/L malt extract, 2.0g/L peptone, g/L yeast extract, 1.0g/L potassium chloride (KCl) 2 ) 0.3g/L, magnesium sulfate heptahydrate (MgSO 4 .7H 2 O) 0.3g/L, monopotassium phosphate (KH) 2 PO 4 ）0.3g/L；
Adding 30% of the volume of a triangular flask into a 100 ml triangular flask, inoculating 1 inclined plane strain to each triangular flask, placing the inoculated strain into a full-temperature shaking incubator at 25 ℃ and a rotating speed of 150 rpm, and culturing for 3d to obtain a secondary strain;
(1.3) three-stage Strain culture
Inoculating the second-level strain to a 5L fermentation tank, wherein the liquid loading amount is 50% of the tank volume, the inoculation amount is 3%, the ventilation is 1:1 (v/v) according to the volume ratio, the pressure is 0.1MP, and the constant-temperature ventilation culture is carried out for 2 days at 25 ℃ to obtain a third-level strain;
the culture medium in the fermentation tank is the same as the liquid shake flask culture medium.
(1.4) four-stage culture
The four-stage culture adopts liquid culture: inoculating the three-level strain to a 50L fermentation tank, wherein the liquid loading amount is 50% of the tank volume, the inoculation amount is 3%, the ventilation is 1:1 (v/v) according to the volume ratio, the pressure is 0.1MP, and the constant-temperature ventilation culture is carried out for 5 days at 25 ℃ to obtain the final fermentation product of the liquid; the culture medium in the fermentation tank is the same as the liquid shake flask culture medium;
(2) Extraction and refinement of active principles in the final fermentation
(2.1) pretreatment of the final fermentation product
Filtering the final fermentation product of the liquid with 0.20 μm filter membrane to obtain mycelium; freeze drying mycelium, and pulverizing to 20 mesh.
(2.2) extraction
Extracting the dried mycelia with methanol; the feed-liquid ratio is 1:2 (W: V), the ultrasonic power is 10KHz, and the extraction time is 10 minutes; centrifuging at 2000 rpm after extraction, concentrating the supernatant at 100deg.C to 1/2 of original volume to obtain concentrated dealcoholized solution, recovering methanol, and refining the concentrated dealcoholized solution by further chromatography.
(2.3) separation and purification
Purification was performed by reverse phase chromatography. The stationary phase is bonding phase filler, the bonding phase filler is reversed-phase carbon-eighteen filler (RPC 18), the particle diameter is 3 microns, and the diameter of the chromatographic column and the flow rate of the mobile phase are determined according to the production scale; ethanol or methanol and water are used according to the proportion of 0 to 100: performing gradient elution according to the proportion of 100-0, and collecting a target peak with the molecular weight of 676 on a mass spectrum detector; concentrating the collected matter below 100deg.C to viscous state, and drying at 170deg.C; the cyclic-threo-valo-isoleucyl-leucinide was obtained as a white powder.
The structural identification result shows that the structural formula of the ring color-threo-valyl-isoleucyl-leucinyl peptide is as follows:
the activity measurement results show that the half-scavenging concentration (DC) of cyclophosphazene-threo-valyl-isoleucyl-leucinide to diphenyl picrylhydrazine free radical (DPPH) 50 ) The method comprises the following steps: 1.31 mg/mL; the diameter of the inhibition zone for Streptomyces albus was 13.74. 13.74mm at a concentration of 200. Mu.g/mL.
Example 2
The preparation of the cyclic color-threo-valyl-isoleucyl-leucinyl peptide with antifungal and free radical scavenging activities is carried out as follows:
the frog manure mould used in this example 2 is a schizophreniform;
(1) Strain culture
The culture method is liquid culture, and comprises the following specific procedures:
(1.1) slant culture of bacterial species
Inoculating the rana chensinensis faecal fungus strain into potato agar (PDA) slant culture medium, and culturing at 36 deg.C for 8d to obtain first-class strain;
(1.2) culturing of secondary strains
Inoculating the first-level strain to a liquid shake flask culture medium for culture;
liquid shake flask medium: glucose 50.0 g/L, malt extract 30.0g/L, peptone 6.0 g/L, yeast extract 4.0g/L, potassium chloride (KCl) 2 ) 0.3g/L, magnesium sulfate heptahydrate (MgSO 4 .7H 2 O) 0.3g/L, monopotassium phosphate (KH) 2 PO 4 ）2.0 g/L。
Adding 50% of the volume of the triangular flask into a 1000 ml triangular flask, inoculating 3 inclined plane strains into each triangular flask, placing the triangular flask into a full-temperature shaking incubator at 36 ℃ and a rotating speed of 250 rpm after inoculation, and culturing for 7d to obtain a secondary strain.
(1.3) three-stage Strain culture
Inoculating the second-level strain into a 50L fermentation tank, wherein the liquid loading amount is 80% of the tank volume, the inoculation amount is 10%, the aeration amount is 1:2 (v/v) by volume ratio, the pressure is 0.2MP, and the constant-temperature aeration culture is carried out for 5 days at 36 ℃ to obtain the third-level strain.
The culture medium in the fermentation tank is the same as the liquid shake flask culture medium.
(1.4) four-stage culture
The four-stage culture is liquid culture: inoculating the three-level strain to a fermentation tank with the volume of more than or equal to 500L, wherein the liquid loading amount is 80% of the tank volume, the inoculum size is 10%, the aeration amount is 1:2 (v/v) in volume ratio, the pressure is 0.2MP, and the constant-temperature aeration culture is carried out for 15 days at 36 ℃ to obtain the final fermentation product of the liquid; the culture medium in the fermentation tank is the same as the liquid shake flask culture medium.
(2) Extraction and refinement of active principles in the final fermentation
(2.1) pretreatment of the final fermentation product
Filtering the final fermentation product of the liquid with a 1.0 μm filter membrane to obtain mycelium; freeze drying mycelium, and pulverizing to 120 mesh.
(2.2) extraction
Extracting the dried mycelium with ethanol at a feed-liquid ratio of 1:20 (W: V) and ultrasonic power of 100KHz for 200 min; centrifuging at 12000 rpm after extraction, concentrating supernatant at 40deg.C to 1/10 of original volume to obtain concentrated dealcoholized solution, recovering ethanol, and refining by chromatography.
(2.3) separation and purification
Purification was performed by reverse phase chromatography. The stationary phase is bonding phase filler, the bonding phase filler is reversed-phase carbon-eighteen filler (RPC 18), the particle diameter is 50 microns, and the diameter of the chromatographic column and the flow rate of the mobile phase are determined according to the production scale; methanol and water are used according to the proportion of 0 to 100: performing gradient elution according to the proportion of 100-0, and collecting a target peak with the molecular weight of 676 on a mass spectrum detector; concentrating the collected matter below 30deg.C to viscous state, and drying at-30deg.C; the cyclic-threo-valo-isoleucyl-leucinide is obtained as a white powder.
The structural identification result shows that the structural formula of the cyclophospha-threo-valyl-isoleucyl-leucinyl peptide is as follows:
the activity measurement results show that the half-scavenging concentration (DC) of cyclophosphazene-threo-valyl-isoleucyl-leucinide to diphenyl picrylhydrazine free radical (DPPH) 50 ) The method comprises the following steps: 1.31 mg/mL; the diameter of the inhibition zone for Streptomyces albus was 13.74. 13.74mm at a concentration of 200. Mu.g/mL.
Example 3
The preparation of the cyclic color-threo-valyl-isoleucyl-leucinyl peptide with antifungal and free radical scavenging activities is carried out as follows:
the frog-faecal mould selected in this example 3 was a solid spore frog-faecal mould.
(1) Strain culture
The culture method is liquid-solid mixed culture, and comprises the following specific procedures:
(1.1) slant seed culture
Inoculating a solid frog faecal fungus strain into a potato agar (PDA) slant culture medium, and culturing for 6d at a constant temperature of 30 ℃ to obtain a first-class strain;
(1.2) culturing of secondary strains
Inoculating the first-level strain to a liquid shake flask culture medium for culture;
liquid shake flask medium: glucose 35.0 g/L, malt extract 20.0 g/L, peptone 4.0g/L, yeast extract 2.0g/L, potassium chloride (KCl) 2 ) 0.3g/L, magnesium sulfate heptahydrate (MgSO 4 .7H 2 O) 0.3g/L, monopotassium phosphate (KH) 2 PO 4 ）1.0 g/L。
Adding 40% of liquid culture medium into 500 ml triangular flasks, inoculating 2 slant strains into each triangular flask, placing into a full-temperature shaking incubator at 30deg.C and rotation speed of 200 rpm, and culturing for 5d to obtain secondary strain.
(1.3) three-stage Strain culture
Inoculating the second-level strain into a 50L fermentation tank, wherein the liquid loading amount is 60% of the tank volume, the inoculation amount is 6%, the aeration amount is 1:1.5 (v/v) according to the volume ratio, the pressure is 0.15MP, and the constant-temperature aeration culture is carried out for 3 days at 30 ℃ to obtain the third-level strain.
The culture medium in the fermentation tank is the same as the liquid shake flask culture medium.
(1.4) four-stage culture
The four-stage culture is solid culture: mixing the three-stage strain into a solid culture medium, wherein the inoculum size is 3% of the solid material, and carrying out aeration culture at a constant temperature of 25 ℃ for 10 days to obtain a solid final fermentation product; the solid culture medium is as follows: glucose 25.0g/L, malt extract 10.0g/L, peptone 2.0g/L, yeast extract 1.0g/L, potassium chloride (KCl) 2 ) 0.3g/L, magnesium sulfate heptahydrate (MgSO 4 .7H 2 O) 0.3g/L, monopotassium phosphate (KH) 2 PO 4 ）0.3g/L; agar 15.0 g/L.
(2) Extraction and refinement of active principles in the final fermentation
(2.1) pretreatment of the final fermentation product
The final fermentation of the solids was dried at 30 ℃ to a water content equal to 20% and then crushed to 20 mesh for use.
(2.2) extraction
Extracting the dried and crushed final fermentation product with ethanol; the feed-liquid ratio is 1:2 (W: V), the ultrasonic power is 10KHz, and the extraction time is 10 minutes; filtering with 0.20 μm filter membrane after extraction; concentrating the filtrate to 1/2 of the original volume at 30deg.C to obtain concentrated dealcoholized solution, recovering ethanol, and refining the concentrated dealcoholized solution by further chromatography;
(2.3) separation and purification
Purification was performed by reverse phase chromatography. The stationary phase is bonding phase filler, the bonding phase filler is reversed-phase carbon-eighteen filler (RPC 18), the particle diameter is 25 micrometers, and the diameter of the chromatographic column and the flow rate of the mobile phase are determined according to the production scale; methanol and water are used according to the proportion of 0 to 100: performing gradient elution according to the proportion of 100-0, and collecting a target peak with the molecular weight of 676 on a mass spectrum detector; concentrating the collected matter at below 40deg.C to viscous state, and drying at 10deg.C; the cyclic-threo-valo-isoleucyl-leucinide is obtained as a white powder.
The structural identification result shows that the structural formula of the cyclophospha-threo-valyl-isoleucyl-leucinyl peptide is as follows:
the activity measurement results show that the half-scavenging concentration (DC) of cyclophosphazene-threo-valyl-isoleucyl-leucinide to diphenyl picrylhydrazine free radical (DPPH) 50 ) The method comprises the following steps: 1.31 mg/mL; the diameter of the inhibition zone for Streptomyces albus was 13.74. 13.74mm at a concentration of 200. Mu.g/mL.
Example 4:
the preparation of the cyclic color-threo-valyl-isoleucyl-leucinyl peptide with antifungal and free radical scavenging activities is carried out as follows:
the frog-excrement mould used in this example 4 is frog-raw frog-excrement mould.
(1) Strain culture
The culture method is liquid-solid mixed culture, and comprises the following specific procedures:
(1.1) slant seed culture
Inoculating the preserved frog-faecal fungus strain into potato agar (PDA) slant culture medium, and culturing at 29 deg.C for 5d to obtain first-class strain;
(1.2) culturing of secondary strains
Inoculating the first-level strain to a liquid shake flask culture medium for culture;
liquid shake flask medium: glucose 40.0 g/L, malt extract 25.0g/L, peptone 3.0g/L, yeast extract 3.0g/L, potassium chloride (KCl) 2 ) 0.3g/L, magnesium sulfate heptahydrate (MgSO 4 .7H 2 O) 0.3g/L, monopotassium phosphate (KH) 2 PO 4 ）1.2 g/L。
Adding 40% of liquid culture medium into 500 ml triangular flasks, inoculating 1 inclined plane strain into each triangular flask, placing into a full-temperature shaking incubator at 30deg.C and rotation speed of 180 rpm, and culturing for 4d to obtain secondary strain.
(1.3) three-stage Strain culture
Inoculating the second-level strain into a 30L fermentation tank, wherein the liquid loading amount is 65% of the tank volume, the inoculation amount is 6%, the aeration amount is 1:1.2 (v/v) according to the volume ratio, the pressure is 0.12MP, and the constant-temperature aeration culture is carried out for 3 days at 32 ℃ to obtain the third-level strain.
The culture medium in the fermentation tank is the same as the liquid shake flask culture medium.
(1.4) four-stage culture
The four-stage culture adopts solid culture: mixing the three-stage liquid strain into a solid culture medium, wherein the inoculation amount is 10% of the solid material amount, and carrying out aeration culture at a constant temperature of 36 ℃ for 8 days to obtain a solid final fermentation product;
the solid culture medium is as follows: glucose 50.0 g/L, malt extract 30.0g/L, peptone 6.0 g/L, yeast extract 4.0g/L, potassium chloride (KCl) 2 ) 0.3g/L, magnesium sulfate heptahydrate (MgSO 4 .7H 2 O) 0.3g/L, monopotassium phosphate (KH) 2 PO 4 ) 2.0. 2.0g/L, 30.0g/L of agar.
(2) Extraction and refinement of active principles in the final fermentation
(2.1) pretreatment of the final fermentation product
The final fermentation product of the solid is dried to water content of 10% at 130 ℃, and then crushed to 120 meshes for standby.
(2.2) extraction
Extracting the dried and crushed final fermentation product with methanol at a feed-liquid ratio of 1:20 (W: V) and ultrasonic power of 100KHz for 200 min; filtering with 1.0 μm filter membrane after extraction; concentrating the filtrate at 40deg.C to 1/10 of the original volume to obtain concentrated dealcoholized solution, recovering methanol, and refining the concentrated dealcoholized solution by further chromatography.
(2.3) separation and purification
Purification was performed by reverse phase chromatography. The stationary phase is bonding phase filler, the bonding phase filler is reversed-phase carbon-eighteen filler (RPC 18), the particle diameter is 10 micrometers, and the diameter of the chromatographic column and the flow rate of the mobile phase are determined according to the production scale; methanol and water are used according to the proportion of 0 to 100: performing gradient elution according to the proportion of 100-0, and collecting a target peak with the molecular weight of 676 on a mass spectrum detector; concentrating the collected matter below 100deg.C to viscous state, and drying at 170deg.C; the cyclic-threo-valo-isoleucyl-leucinide is obtained as a white powder.
The structural identification result shows that the structural formula of the cyclophospha-threo-valyl-isoleucyl-leucinyl peptide is as follows:
the activity measurement results show that the half-scavenging concentration (DC) of cyclophosphazene-threo-valyl-isoleucyl-leucinide to diphenyl picrylhydrazine free radical (DPPH) 50 ) The method comprises the following steps: 1.31 mg/mL; the diameter of the inhibition zone for Streptomyces albus was 13.74. 13.74mm at a concentration of 200. Mu.g/mL.
Claims (2)
1. A cyclic-color-threo-valyl-isoleucyl-leucinyl peptide having antifungal and free radical scavenging activity characterized by: the structural formula of the cyclic-color-threo-valyl-isoleucyl-leucinyl peptide is as follows:
；
the cyclic color-threo-valyl-isoleucyl-leupeptin is fifteen-membered cyclic pentapeptide formed by connecting tryptophan, threonine, valine, isoleucine and leucine through an amide bond, and is white powder;
the cyclophosphamide-threo-valyl-isoleucyl-leupeptin has free radical scavenging activity and anti-streptoverticillium albuminosus activity;
half-value scavenging concentration DC of p-diphenyl picrylphenylhydrazine free radical DPPH 50 The method comprises the following steps: the diameter of the inhibition zone for Streptomyces albus at a concentration of 200. Mu.g/mL was 13.74mm, which was 1.31 mg/mL.
2. A process for the preparation of cyclic-color-threo-valyl-isoleucyl-leucinyl peptides having antifungal and free radical scavenging activity as claimed in claim 1, characterized by the following operative steps:
(1) Strain culture
The frog manure mould is preparedBasidiobolussp.) carrying out slant strain culture, secondary strain culture, tertiary strain culture and quaternary culture to obtain a liquid final fermentation product or a solid final fermentation product;
(2) Extraction and refinement of active principles in the final fermentation
Extracting the effective components in the final fermentation product with methanol or ethanol, and purifying by reverse phase chromatography to obtain white powdery cyclophosphamide-threo-valyl-isoleucyl-leucinyl peptide;
the frog manure mould is preparedBasidiobolussp.) is one of frog-derived frog-feces mould, solid spore frog-feces mould or schizospore frog-feces mould;
the specific operation of the step (1) is as follows:
(1.1) slant culture of bacterial species
Inoculating the frog-fecal mould strain into potato agar PDA slant culture medium, and culturing for 4-8 d at constant temperature of 25-36 ℃ to obtain slant strain;
(1.2) culturing of secondary strains
Inoculating the slant strain to a liquid shake flask culture medium for culture;
adding a liquid culture medium with the volume of 30-50% of that of a triangular flask in the triangular flask with the volume of more than or equal to 100 ml, inoculating 1-3 inclined plane strains to each triangular flask, and culturing for 3-7 d in a full-temperature shaking incubator at the temperature of 25-36 ℃ and the rotating speed of 150-250 rpm to obtain a secondary strain;
the liquid shake flask culture medium is prepared from 25.0-50.0 g/L glucose, 10.0-30.0 g/L malt extract, 2.0-6.0 g/L peptone, 1.0-4.0 g/L yeast extract powder, 0.3-2.0 g/L potassium chloride, 0.3-2.0 g/L magnesium sulfate heptahydrate, 0.3-2.0 g/L potassium dihydrogen phosphate and water;
(1.3) three-stage Strain culture
Inoculating the second-level strain to a fermentation tank with the volume of more than or equal to 5L, wherein the liquid loading amount is 50-80% of the tank volume, the inoculation amount is 3-10%, and carrying out constant-temperature aeration culture for 2-5 days under the conditions that the aeration rate is 1:1-2 (v/v), the pressure is 0.1-0.2 MP and the temperature is 25-36 ℃ to obtain a third-level strain;
the medium in the fermenter is the liquid medium in step (1.2);
(1.4) four-stage culture
The four-stage culture is liquid culture, three-stage strains are inoculated into a fermentation tank with the volume of more than or equal to 50L, the liquid loading amount is 50-80 percent of the volume of the tank, the inoculum size is 3-10 percent, and the final fermentation product of the liquid is obtained by constant-temperature aeration culture for 5-15 days under the conditions that the aeration rate is 1:1-2 (v/v), the pressure is 0.1-0.2 MP and the temperature is 25-36 ℃; the medium in the fermenter is the liquid shake flask medium in step (1.2);
or, the four-stage culture is solid culture, three-stage liquid strains are mixed into a solid culture medium, the inoculation amount is 3-10% of the solid material amount, and the constant temperature culture is carried out for 5-15 days at 25-36 ℃ to obtain a solid final fermentation product;
the solid culture medium is prepared by uniformly mixing 25.0-50.0 g/L of glucose, 10.0-30.0 g/L of malt extract, 2.0-6.0 g/L of peptone, 1.0-4.0 g/L of yeast extract powder, 0.3-2.0 g/L of potassium chloride, 0.3-2.0 g/L of magnesium sulfate heptahydrate, 0.3-2.0 g/L of potassium dihydrogen phosphate, 15.0-30.0 g/L of agar and water;
the specific operation of the step (2) is as follows:
(2.1) pretreatment of the final fermentation product
Filtering the final fermentation product of the liquid by a 0.20-1.0 mu m filter membrane or centrifuging at 2000-12000 r/min to obtain mycelium; freeze drying mycelium, crushing, and sieving with a 20-120 mesh sieve to obtain a pretreated substance;
or, drying the solid final fermentation product at 30-130 ℃ until the water content is less than or equal to 20%, and then crushing to obtain a 20-120-mesh pretreatment product;
(2.2) extraction, separation and purification of the active ingredient in the pretreated matter
(2.2.1) extraction
Extracting the pretreated material with methanol or ethanol; the ratio of the feed to the liquid is 1:2-20 (W: V), the ultrasonic power is 10-100 KHz, and the extraction time is 10-200 minutes; centrifuging at a rotation speed of 2000 rpm or more, or filtering with a 0.20-1.0 μm filter membrane; concentrating the centrifugal supernatant or filtrate to 1/2-1/10 of the original volume at a temperature of less than or equal to 100 ℃ to obtain a concentrated dealcoholized liquid, and simultaneously recovering methanol or ethanol, wherein the concentrated dealcoholized liquid is used for further chromatographic refining;
(2.2.2) separation and purification
Purifying by adopting a reversed phase chromatography method, wherein the stationary phase is a bonding phase filler, the bonding phase filler is reversed phase carbon eighteen filler (RPC 18), and the particle diameter is 3-50 microns; methanol and water are used according to the proportion of 0 to 100: gradient elution is carried out according to the proportion of 100-0, and chromatographic fractions with the molecular weight of 676Da on a mass spectrum detector are collected; concentrating the collected matter at 100 deg.c or lower to viscous state, and drying at 170 deg.c or lower; the cyclic-threo-valo-isoleucyl-leucinide is obtained as a white powder.
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