CN113264987B - Cyclochrome-threo-valerine-isoleutin-leupeptide with antifungal and free radical scavenging activity and preparation method thereof - Google Patents

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

cyclochrome-threonine-valerine-isoleutin-leupeptide with antifungal and free radical scavenging activity and its preparation Preparation method

Technical field

The invention belongs to the technical field of extraction and preparation of biological products, and specifically relates to the extraction and purification of Trp-Thr-Val-Ile-Leu cyclic peptide with antifungal and free radical scavenging activity. .

Background technique

Cyclochrome-threo-valerin-isoleukin-leupeptide is a bioactive substance with antifungal and free radical scavenging activity isolated and prepared from a culture of Basidiobolus sp.

Basidiobolus sp. is a fungus of the genus Basidiobolus in the order Basidiobolus, family Basidiobolus; the common ones in China include Basidiobolus sp., Basidiobolus sp., and Schizospermum sp. The above bacterial species can be isolated from soil and amphibian feces.

Antifungal activity: There are many kinds of fungi that are opportunistic pathogens. People are prone to infection when their resistance is reduced, especially the elderly and patients are prone to fungal infection. At present, the world is aging increasingly. At the same time, due to factors such as environmental pollution, people's resistance is on a downward trend, fungal infection cases are on an upward trend, and resistance to early antifungal drugs has emerged. Therefore, it is very meaningful to develop new antifungal active substances. .

Free radical scavenging activity: Free radicals refer to groups, atoms or molecules containing unpaired electrons from a chemical structure perspective. Free radicals are highly chemically reactive. For living organisms, free radicals are intermediate products of various biochemical reactions in life activities. Under normal circumstances, free radicals in the human body are in a dynamic balance of production and elimination. Free radicals are an effective defense system of the body. Failure to maintain a certain level of free radicals will have adverse effects on the body's life activities. However, free radicals are produced too much or are cleared too slowly. By attacking macromolecules of life and various organelles, they can cause various damages at the molecular level, cellular level, and tissue and organ levels of the body, accelerate the aging process of the body, and induce various diseases. . The latest research shows that human aging and many diseases are related to free radical damage. Substances with free radical scavenging activity can react with free radicals and reduce them to non-free radical compounds. They can scavenge excessive free radicals produced during the metabolism of the body and are important active substances that can improve human health. Free radical scavengers have antioxidant effects in non-living systems, can effectively prevent the oxidative deterioration of substances, and play an important role in extending the shelf life of items. They are widely used in food, medicine, daily chemicals, etc.

Contents of the invention

The object of the present invention is to provide cyclochrom-threo-valerine-isoleutin-leupeptide with antifungal and free radical scavenging activity and a preparation method thereof.

In order to find new natural and highly efficient substances with antifungal activity and free radical scavenging activity, the inventors conducted activity studies on the metabolites of more than 100 entomopathogenic fungi in China and discovered the cyclochrome- Su-Val-Isoleucine-Leuptide has strong anti-Streptococcus albicans activity and free radical scavenging activity.

The structural formula of cyclochrom-threo-valerin-isoleutin-leuptide with antifungal and free radical scavenging activity is as follows:

The cyclochromo-threonine-valerine-isoleucine-leupeptide is a fifteen-membered cyclic five-membered peptide composed of tryptophan, threonine, valine, isoleucine and leucine, connected by amide bonds. Peptide is a white powder;

The cyclochrome-threo-valerian-isoleutin-leupeptide has free radical scavenging activity and anti-Streptococcus albicans activity;

The half scavenging concentration (DC ₅₀ ) of p-diphenylpicrylphenylhydrazyl free radical (DPPH) is: 1.31mg/mL. At a concentration of 200 μg/mL, the diameter of the inhibition zone against Streptococcus albicans is 13.74mm.

The preparation steps of cyclochrom-threo-valerin-isoleutin-leuptide with antifungal and free radical scavenging activity are as follows:

(1) Bacterial culture

Pass Basidiobolus sp. through slant culture, secondary culture, third-level culture and fourth-level culture to obtain a liquid final fermentation product or a solid final fermentation product;

The said Basidiobolus sp. is one of Basidiobolus sp., Basidiobolus sp., or Basidiobolus sp.

(2) Extraction and purification of active ingredients in the final fermentation product

The active ingredients in the final fermentation product are extracted with methanol or ethanol, and purified using reversed-phase chromatography to obtain white powdery cyclochromo-threo-valerin-isoleutin-leupeptide.

The further limited preparation technical scheme is as follows:

The specific operations of step (1) are as follows:

(1.1) Slant culture

Inoculate the frog feces mold strain into potato agar (PDA) slant medium, culture it at a constant temperature of 25 to 36°C for 4 to 8 days, and obtain the first-level strain;

(1.2) Secondary strain culture

Inoculate the primary bacterial strain into the liquid shake flask culture medium;

Add 30 to 50% of the volume of the Erlenmeyer flask to a 100 ml Erlenmeyer flask. Inoculate 1 to 3 slant bacterial strains into each Erlenmeyer flask. In a full-temperature shaking incubator, the temperature is 25-36°C and the rotation speed is 150. Under the condition of ~250 rpm, cultivate for 3 to 7 days to obtain the secondary bacterial strain;

The liquid shake flask culture medium consists of glucose 25.0~50.0 g/L, malt extract 10.0~30.0g/L, peptone 2.0~6.0 g/L, yeast extract powder 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, potassium dihydrogen phosphate (KH ₂ PO ₄ ) 0.3 ~ 2.0 g/L;

(1.3) Third-level bacterial culture

Inoculate the secondary bacteria into a fermentation tank larger than or equal to 5L. The liquid filling volume is 50-80% of the tank volume, the inoculation volume is 3-10%, and the ventilation volume is 1:1-2 (v/v), Under the conditions of pressure 0.1~0.2MP and 25~36℃, cultivate with constant temperature ventilation for 2~5 days to obtain the third-level bacterial strain;

The culture medium in the fermentor is the same as the liquid culture medium in step (1.2);

(1.4) Fourth-level training

The fourth-level culture is liquid culture. The third-level bacteria are inoculated into a fermentation tank of 50L or larger. The liquid volume is 50-80% of the tank volume, the inoculum volume is 3-10%, and the ventilation volume is 1:1~ 2 (v/v), pressure 0.1~0.2MP, 25~36℃, constant temperature ventilation culture for 5~15 days to obtain the final liquid fermentation product; the liquid culture medium in the culture medium step (1.2) in the fermentor .

In the step (1.4), the fourth-level culture is solid culture, and the third-level liquid strain is mixed into the solid culture medium. The inoculum amount is 3 to 10% of the solid material amount, and the culture is carried out at a constant temperature of 25 to 36°C for 5 to 15 days. , to obtain a solid final fermentation product; 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 powder 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, potassium dihydrogen phosphate (KH ₂ PO ₄ ) 0.3～2.0 g/L, Made by mixing agar 15.0~30.0 g/L and water evenly.

The specific operations of step (2) are as follows:

(2.1) Pretreatment of final fermentation product

Filter the final fermentation product of the liquid through a 0.20-1.0 μm filter membrane or centrifuge at 2000-12000 rpm to obtain mycelium; freeze-dry the mycelium and crush it to obtain a pretreated product that passes through a 20-120 mesh sieve;

Or, dry the solid final fermentation product at 30-130°C until the moisture content is less than or equal to 20%, and then crush it into a pre-processed product of 20-120 mesh;

(2.2) Extraction, separation and purification of active ingredients in pretreatment materials

(2.2.1) Extraction

Extract the pretreated material with methanol or ethanol; the material-liquid ratio is 1:2~20 (W:V), the ultrasonic power is 10~100KHz, the extraction time is 10~200 minutes; centrifuge at a speed of 2000 rpm or greater. , or 0.20~1.0μm filter membrane filtration; take the filtrate or centrifugal supernatant, and concentrate it to 1/2~1/10 of the original volume at a temperature of 100°C or less to obtain a concentrated dealcoholization liquid, while recovering methanol or ethanol, concentrating and de-alcoholizing. The alcohol liquid is used for further chromatographic purification;

(2.2.2) Separation and purification

Reverse-phase chromatography is used for purification. The stationary phase is a bonded phase filler. The bonded phase filler is a reverse-phase carbon eighteen filler, that is, RPC18. The particle diameter is 3 to 50 microns; use methanol and water at a ratio of 0 to 100:100 to 0 ratio for gradient elution, collect the chromatographic fraction with a molecular weight of 676 on the mass spectrometer detector; concentrate the collection to a viscous state at a temperature of less than or equal to 100°C, and dry it at a temperature lower than 170°C; obtain a white powder The cyclic color-threonine-valerine-isoleutin-leutin.

The analytical research description of the present invention is as follows:

1. Screening research found that a methanol extract of Basidiobolus sp. mycelium has strong free radical scavenging activity and anti-Streptococcus albicans activity:

(1) Determination of the inhibitory activity of methanol extract against Streptococcus albicans: Prepare an extract concentration of 1.0 mg/ml 20% DMSO solution, use amphotericin B as a positive control, and use 20% DMSO solution as a blank control for inhibition. In the Streptococcus albicans test, the average inhibition zone diameter of the extract against Streptococcus albicans was calculated to be 11.53 mm.

(2) Measurement of free radical scavenging activity of methanol extract: Prepare methanol solutions with extract sample concentrations of 0.2, 0.4, 0.8, 1.0, and 1.6 mg/ml, and measure its activity against 0.5 mmol/L DPPH free radical at 517 nm. Scavenging activity, the half scavenging concentration of DPPH free radicals was calculated to be 1.49 mg/mL.

Since the content of active ingredients in the methanol extract is low, further extraction and purification of the active ingredients are required.

2. The methanol extract was further separated and purified using reversed-phase chromatography to obtain a pure product with the following biological activities:

(1) Determination of the inhibitory activity of cyclochrome-threonine-valerine-isoleukin-leupeptide against Streptococcus albicans: Prepare an extract concentration of 0.2 mg/ml 20% DMSO solution, use amphotericin B as a positive control, and use 20 % DMSO solution was used as a blank control, and the test for inhibiting Streptococcus albicans was carried out. The average inhibition zone diameter of the extract against Streptococcus albicans was calculated to be 13.74 mm.

(2) Determination of free radical scavenging activity of cyclochrome-threo-valerine-isoleutin-leuptide: Prepare extract sample concentrations of 0.2, 0.4, 0.8, 1.0, 1.6 mg/ml methanol solution, and measure its effect at 517 nm The DPPH free radical scavenging activity of 0.5 mmol/L was calculated to have a half scavenging concentration of DPPH free radical of 1.31 mg/mL.

3. Identification of chemical structure of active compounds

High-resolution liquid mass spectrometry analysis shows that the purified active compound has positive ions of 585.3440 (M+H ⁺ ), 607.3261 (M+Na ⁺ ), and 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 table below:

Comprehensive liquid mass spectrometry analysis and nuclear magnetic resonance analysis revealed that the structure of the isolated and purified active compound is cyclochrom-threo-valerine-isoleutin-leuptide, as shown in the above chemical structural formula.

The beneficial technical effects of the present invention are reflected in the following aspects:

1. The cyclochrome-threo-valerine-isoleutin-leupeptide prepared by the method of the present invention has the activity of inhibiting Streptococcus albicans and scavenging free radicals, and opens up a new application field of Fructus faecalis. The present invention discovers for the first time that the fungus Frugospora has the function of metabolizing the above-mentioned active substances. Based on this invention, it is expected to further clone relevant genes and construct high-yield strains.

2. Cyclochrome-threo-valerine-isoleutin-leuptide is a new type of framework that can be used as a drug lead compound for derivatization to create more active compounds.

3. The cyclochrome-threo-valerine-isoleutin-leupeptide of the present invention can be used to prepare Streptococcus albicans inhibitors and free radical scavengers. Free radical scavengers often have functions such as whitening, antioxidant, anti-aging, preservation, sterilization, and anti-inflammatory. Streptococcus albicans inhibitors prepared from cyclochrome-threonine-valerine-isoleutin-leuptide can prevent and treat fungal infections.

4. Since the present invention adopts microbial fermentation production, it is not affected by the environment and resources, and can easily realize industrialization and automation, and is not affected by the environment and natural resources.

5. The products produced according to the process of the present invention have low cost, simple process, stable process, easy control and high success rate.

Detailed ways

The present invention will be further described below in conjunction with examples.

Example 1:

The preparation steps of a cyclochrom-threo-valerin-isoleutin-leuptide with antifungal and free radical scavenging activity are as follows:

The fungus used in Example 1 is Frogspora frog.

(1) Strain culture

(1.1) Inclined seed culture

Inoculate the Frognata faecalis strain into potato agar (PDA) slant culture medium, culture it at a constant temperature of 25°C for 4 days, and obtain the first-level strain;

(1.2) Secondary strain culture

Inoculate the primary bacterial strain into the liquid shake flask culture medium;

Liquid shake flask culture medium: glucose 25.0g/L, malt extract 10.0g/L, peptone 2.0g/L, yeast extract powder 1.0g/L, potassium chloride (KCl ₂ ) 0.3g/L, magnesium sulfate heptahydrate (MgSO ₄ .7H ₂ O) 0.3g/L, potassium dihydrogen phosphate (KH ₂ PO ₄ ) 0.3g/L;

Add 30% of the volume of the flask to a 100 ml Erlenmeyer flask. Inoculate 1 slant bacterial strain into each Erlenmeyer flask. After inoculation, place it in a full-temperature shaking incubator at a temperature of 25°C and a rotation speed of 150 rpm. Cultivate for 3 days, that is, Obtain secondary strains;

(1.3) Third-level bacterial culture

Inoculate the secondary bacteria into the 5L fermentation tank. The liquid volume is 50% of the tank volume, the inoculum volume is 3%, the ventilation volume is 1:1 (v/v), the pressure is 0.1MP, and the constant temperature ventilation culture is 25°C. In 2 days, third-level strains were obtained;

The culture medium in the fermentor is the same as the liquid shake flask culture medium.

(1.4) Fourth-level training

The fourth-level culture uses liquid culture: inoculate the third-level bacteria into a 50L fermentation tank, the liquid volume is 50% of the tank volume, the inoculum volume is 3%, the ventilation volume is 1:1 (v/v), and the pressure is 0.1 MP, culture at 25°C for 5 days with constant temperature ventilation to obtain the final liquid fermentation product; the culture medium in the fermentation tank is the same as the liquid shake flask culture medium;

(2) Extraction and purification of active ingredients in the final fermentation product

(2.1) Pretreatment of final fermentation product

The final fermentation product of the liquid was filtered through a 0.20 μm filter membrane to obtain mycelium; the mycelium was freeze-dried and crushed to 20 mesh for later use.

(2.2) Extraction

Extract the aforementioned dried mycelium with methanol; the material-to-liquid ratio is 1:2 (W:V), the ultrasonic power is 10KHz, and the extraction time is 10 minutes; after the extraction is completed, centrifuge at a speed of 2000 rpm, and the centrifugal supernatant is Concentrate to 1/2 of the original volume at a temperature of 100°C to obtain a concentrated dealcoholized liquid. Methanol is recovered at the same time, and the concentrated dealcoholized liquid is used for further chromatographic purification.

(2.3) Separation and purification

Purified using reverse phase chromatography. The stationary phase is a bonded phase filler. The bonded phase filler is a reversed-phase carbon eighteen filler, that is, RPC18. The particle diameter is 3 microns. The diameter of the chromatographic column and the flow rate of the mobile phase can be determined according to the production scale; ethanol or methanol and water are used. Carry out gradient elution according to the ratio of 0~100:100~0, and collect the target peak with a molecular weight of 676 on the mass spectrometer detector; the collection is concentrated to a viscous state below 100°C, and dried at 170°C; a white powder is obtained The cyclic color-threonine-valerine-isoleutin-leutin.

The structural identification results show that the structural formula of cyclochrom-threo-valerine-isoleutin-leuptide is as follows:

The activity measurement results show that the half scavenging concentration (DC ₅₀ ) of diphenylpicrylphenylhydrazine free radical (DPPH) of cyclochrome-threo-valerine-isoleutin-leuptide is: 1.31 mg/mL; at 200 μg/mL The diameter of the inhibition zone against Streptococcus albicans is 13.74 mm.

Example 2

The preparation steps of cyclochrom-threo-valerin-isoleutin-leuptide with antifungal and free radical scavenging activity are as follows:

The fungus used in Example 2 is Frugospora;

(1) Strain culture

The culture method is liquid culture, and the specific procedures are as follows:

(1.1) Slant culture

Inoculate the Schizospermum faecalis strain into potato agar (PDA) slant culture medium, culture it at a constant temperature of 36°C for 8 days, and obtain the first-level strain;

(1.2) Secondary strain culture

Inoculate the primary bacterial strain into the liquid shake flask culture medium;

Liquid shake flask culture medium: glucose 50.0 g/L, malt extract 30.0 g/L, peptone 6.0 g/L, yeast extract powder 4.0g/L, potassium chloride (KCl ₂ ) 0.3g/L, magnesium sulfate heptahydrate (MgSO ₄ .7H ₂ O) 0.3g/L, potassium dihydrogen phosphate (KH ₂ PO ₄ ) 2.0 g/L.

Add 50% of the volume of the flask to a 1000 ml Erlenmeyer flask. Inoculate 3 strains of slant bacteria into each Erlenmeyer flask. After inoculation, place it in a full-temperature shaking incubator at a temperature of 36°C and a rotation speed of 250 rpm. Cultivate for 7 days, that is, Obtain secondary strains.

(1.3) Third-level bacterial culture

Inoculate the secondary bacterial strain into a 50L fermentation tank. The liquid volume is 80% of the tank volume, the inoculum volume is 10%, the ventilation volume is 1:2 (v/v), the pressure is 0.2MP, and the constant temperature ventilation culture is 36°C for 5 Days later, a third-level bacterial strain was obtained.

The culture medium in the fermentor is the same as the liquid shake flask culture medium.

(1.4) Fourth-level training

The fourth-level culture is liquid culture: inoculate the third-level bacteria into a fermentation tank larger than or equal to 500L. The liquid volume is 80% of the tank volume, the inoculum volume is 10%, and the aeration volume is based on the volume ratio of 1:2 (v/v). Pressure 0.2MP, constant temperature ventilation culture at 36°C for 15 days to obtain the final liquid fermentation product; the culture medium in the fermentation tank is the same as the liquid shake flask culture medium.

(2) Extraction and purification of active ingredients in the final fermentation product

(2.1) Pretreatment of final fermentation product

The final fermentation product of the liquid was filtered through a 1.0 μm filter membrane to obtain mycelium; the mycelium was freeze-dried and crushed to 120 mesh for later use.

(2.2) Extraction

Extract the aforementioned dried mycelium with ethanol, with a solid-liquid ratio of 1:20 (W:V), an ultrasonic power of 100KHz, and an extraction time of 200 minutes; after the extraction, centrifuge at a speed of 12,000 rpm, and the centrifugal supernatant is at a temperature of Concentrate to 1/10 of the original volume at 40°C to obtain a concentrated dealcoholized liquid. At the same time, ethanol is recovered, and the concentrated dealcoholized liquid is used for further chromatographic purification.

(2.3) Separation and purification

Purified using reverse phase chromatography. The stationary phase is a bonded phase filler. The bonded phase filler is a reversed phase carbon eighteen filler, that is, RPC18. The particle diameter is 50 microns. The diameter of the chromatographic column and the flow rate of the mobile phase can be determined according to the production scale; use methanol and water at 0 Perform gradient elution with a ratio of ~100:100~0, and collect the target peak with a molecular weight of 676 on the mass spectrometer detector; the collection is concentrated to a viscous state below 30°C, and dried at -30°C; a white powder is obtained The cyclic color-threonine-valerine-isoleutin-leutin.

Structural identification results show that the structural formula of cyclochrome-threo-valerine-isoleutin-leuptide is as follows:

The activity measurement results show that the half scavenging concentration (DC ₅₀ ) of cyclochrome-threo-valerine-isoleutin-leuptide on diphenylpicrylphenylhydrazine free radical (DPPH) is: 1.31 mg/mL; at 200 μg/mL The diameter of the inhibition zone against Streptococcus albicans is 13.74 mm.

Example 3

The preparation steps of cyclochrom-threo-valerin-isoleutin-leuptide with antifungal and free radical scavenging activity are as follows:

The mold that was selected in this Example 3 is B. sporogenes.

(1) Strain culture

The culture method is liquid-solid mixed culture, and the specific procedures are as follows:

(1.1) Inclined seed culture

Inoculate the Apodophora faecalis strain into potato agar (PDA) slant culture medium, culture it at a constant temperature of 30°C for 6 days, and obtain the first-level strain;

(1.2) Secondary strain culture

Inoculate the primary bacterial strain into the liquid shake flask culture medium;

Liquid shake flask medium: glucose 35.0 g/L, malt extract 20.0 g/L, peptone 4.0 g/L, yeast extract powder 2.0g/L, potassium chloride (KCl ₂ ) 0.3g/L, magnesium sulfate heptahydrate (MgSO ₄ .7H ₂ O) 0.3g/L, potassium dihydrogen phosphate (KH ₂ PO ₄ ) 1.0 g/L.

Add 40% of the volume of the Erlenmeyer flask to a 500 ml Erlenmeyer flask. Inoculate 2 strains of slant bacteria into each Erlenmeyer flask. After inoculation, place it in a full-temperature shaking incubator at a temperature of 30°C and a rotation speed of 200 rpm. Cultivate for 5 days, that is, Obtain secondary strains.

(1.3) Third-level bacterial culture

Inoculate the secondary bacterial strain into a 50L fermentation tank. The liquid volume is 60% of the tank volume, the inoculum volume is 6%, the ventilation volume is 1:1.5 (v/v), the pressure is 0.15MP, and the constant temperature ventilation culture is 30°C. In 3 days, the third-level bacteria were obtained.

The culture medium in the fermentor is the same as the liquid shake flask culture medium.

(1.4) Fourth-level training

The fourth-level culture is solid culture: mix the third-level bacterial strain into the solid culture medium, the inoculum amount is 3% of the solid material amount, and culture it at 25°C for 10 days with constant temperature ventilation to obtain the solid final fermentation product; the solid culture medium is : Glucose 25.0 g/L, malt extract 10.0 g/L, peptone 2.0 g/L, yeast extract powder 1.0g/L, potassium chloride (KCl ₂ ) 0.3g/L, magnesium sulfate heptahydrate (MgSO ₄ .7H ₂ O) 0.3g/L, potassium dihydrogen phosphate (KH ₂ PO ₄ ) 0.3 g/L; agar 15.0 g/L.

(2) Extraction and purification of active ingredients in the final fermentation product

(2.1) Pretreatment of final fermentation product

The solid final fermentation product is dried at 30°C until the moisture content is equal to 20%, and then crushed to 20 mesh for later use.

(2.2) Extraction

Extract the dried and crushed final fermentation product with ethanol; the material-to-liquid ratio is 1:2 (W:V), the ultrasonic power is 10KHz, and the extraction time is 10 minutes; after the extraction is completed, it is filtered with a 0.20μm filter membrane; the filtrate is concentrated at a temperature of 30°C To 1/2 of the original volume, a concentrated dealcoholization liquid is obtained, ethanol is recovered at the same time, and the concentrated dealcoholization liquid is used for further chromatographic purification;

(2.3) Separation and purification

Purified using reverse phase chromatography. The stationary phase is a bonded phase filler. The bonded phase filler is a reversed-phase carbon eighteen filler, that is, RPC18. The particle diameter is 25 microns. The diameter of the chromatographic column and the flow rate of the mobile phase can be determined according to the production scale; use methanol and water at 0 ~100:100~0 ratio for gradient elution, and the target peak with a molecular weight of 676 on the mass spectrometer detector was collected; the collection was concentrated to a viscous state below 40°C, and dried at 10°C; a white powder was obtained. Cyclose-su-valerium-isoleutin-leutin.

Structural identification results show that the structural formula of cyclochrome-threo-valerine-isoleutin-leuptide is as follows:

The activity measurement results show that the half scavenging concentration (DC ₅₀ ) of diphenylpicrylphenylhydrazine free radical (DPPH) of cyclochrome-threo-valerine-isoleutin-leuptide is: 1.31 mg/mL; at 200 μg/mL The diameter of the inhibition zone against Streptococcus albicans is 13.74 mm.

Example 4:

The preparation steps of cyclochrom-threo-valerin-isoleutin-leuptide with antifungal and free radical scavenging activity are as follows:

The fungus used in Example 4 is Frogspora frog.

(1) Strain culture

The culture method is liquid-solid mixed culture, and the specific procedures are as follows:

(1.1) Inclined seed culture

The preserved frog feces mold strain was inoculated into potato agar (PDA) slant culture medium, and cultured at a constant temperature of 29°C for 5 days to obtain the first-level strain;

(1.2) Secondary strain culture

Inoculate the primary bacterial strain into the liquid shake flask culture medium;

Liquid shake flask medium: glucose 40.0 g/L, malt extract 25.0 g/L, peptone 3.0 g/L, yeast extract powder 3.0g/L, potassium chloride (KCl ₂ ) 0.3g/L, magnesium sulfate heptahydrate (MgSO ₄ .7H ₂ O) 0.3g/L, potassium dihydrogen phosphate (KH ₂ PO ₄ ) 1.2 g/L.

Add 40% of the volume of the flask to a 500 ml Erlenmeyer flask. Inoculate 1 slant bacterial strain into each Erlenmeyer flask. After inoculation, place it in a full-temperature shaking incubator at a temperature of 30°C and a rotation speed of 180 rpm. Cultivate for 4 days, that is, Obtain secondary strains.

(1.3) Third-level bacterial culture

Inoculate the secondary bacteria into a 30L fermentation tank. The liquid volume is 65% of the tank volume, the inoculum volume is 6%, the ventilation volume is 1:1.2 (v/v), the pressure is 0.12MP, and the constant temperature ventilation culture is 32°C. In 3 days, the third-level bacteria were obtained.

The culture medium in the fermentor is the same as the liquid shake flask culture medium.

(1.4) Fourth-level training

The fourth-level culture adopts solid culture: mix the third-level liquid strain into the solid culture medium, the inoculum amount is 10% of the solid material amount, and cultivate it for 8 days at 36°C with constant temperature ventilation to obtain the solid final fermentation product;

The solid culture medium is: glucose 50.0 g/L, malt extract 30.0 g/L, peptone 6.0 g/L, yeast extract powder 4.0g/L, potassium chloride (KCl ₂ ) 0.3g/L, magnesium sulfate heptahydrate ( MgSO ₄ .7H ₂ O) 0.3g/L, potassium dihydrogen phosphate (KH ₂ PO ₄ ) 2.0g/L, agar 30.0 g/L.

(2) Extraction and purification of active ingredients in the final fermentation product

(2.1) Pretreatment of final fermentation product

The solid final fermentation product was dried at 130°C to a moisture content of 10%, and then crushed to 120 mesh for later use.

(2.2) Extraction

Extract the dried and crushed final fermentation product with methanol. The material-to-liquid ratio is 1:20 (W:V), the ultrasonic power is 100KHz, and the extraction time is 200 minutes. After the extraction, it is filtered with a 1.0 μm filter membrane; the filtrate is concentrated at 40°C. To 1/10 of the original volume, a concentrated dealcoholization liquid is obtained, while methanol is recovered, and the concentrated dealcoholization liquid is used for further chromatographic purification.

(2.3) Separation and purification

Purified using reverse phase chromatography. The stationary phase is a bonded phase filler. The bonded phase filler is a reversed-phase carbon eighteen filler, that is, RPC18. The particle diameter is 10 microns. The diameter of the chromatographic column and the flow rate of the mobile phase can be determined according to the production scale; use methanol and water at 0 ~100:100~0 ratio for gradient elution, and the target peak with a molecular weight of 676 on the mass spectrometer detector was collected; the collection was concentrated to a viscous state below 100°C, and dried at 170°C; a white powder was obtained. Cyclose-su-valerium-isoleutin-leutin.

Structural identification results show that the structural formula of cyclochrome-threo-valerine-isoleutin-leuptide is as follows:

The activity measurement results show that the half scavenging concentration (DC ₅₀ ) of cyclochrome-threo-valerine-isoleutin-leuptide on diphenylpicrylphenylhydrazine free radical (DPPH) is: 1.31 mg/mL; at 200 μg/mL The diameter of the inhibition zone against Streptococcus albicans is 13.74 mm.

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.