CN115651067A - Cyclo-C-Val-isoleu-leucin with hepatotoxicity and alpha-glucosidase inhibition activity and preparation method thereof - Google Patents
Cyclo-C-Val-isoleu-leucin with hepatotoxicity and alpha-glucosidase inhibition activity and preparation method thereof Download PDFInfo
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- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention relates to a cyclo-c-v-i-n-Leu peptide (Trp-Ala-Val-Ile-Leu cyclic peptide) with hepatotoxicity and alpha-glucosidase inhibitory activity and a preparation method thereof, belonging to the technical field of extraction and preparation of biological products. The cyclo-Tr-Ala-Val-Ile-Leu peptide is a pentadecatomic cyclic pentapeptide which is formed by connecting tryptophan, alanine, valine, isoleucine and leucine through amide bonds. This is a novel compound and has not been reported in the literature. The compound of the invention can be obtained by culturing ranunculus japonicus and then extracting and separating. The pure product of the cyclo-C-Val-isoleucin is white powder and has half-Inhibitory Concentration (IC) on liver cancer cell HepG2 50 ) Comprises the following steps: 54.19 μ g/mL, half inhibitory concentration of alpha-glucosidase (IC) 50 ) Comprises the following steps: 63.64. Mu.g/mL. The compound can be used as a potential drug for treating liver cancer and weight loss.
Description
Technical Field
The invention belongs to the technical field of extraction and preparation of biological products, and relates to extraction and purification of a Trp-Ala-Val-Ile-Leu cyclopeptide (Trp-Ala-Val-Ile-Leu cyclopeptide) with hepatotoxicity and alpha-glucosidase inhibitory activity.
Background
Cyclochromene-C-valine-isoleucin is extracted from a plant of Rana chensinensis (Va. Laevigata)Basidiobolussp.) separating the obtained bioactive substances with hepatotoxicity and alpha-glucosidase inhibitory activity from the culture.
Botrytis cinerea (A. Frog-dung)Basidiobolussp.) fungi of the genus Rana of the family Ranunculaceae of the order Entomophthorales; common in China are frog-growing frog dung mold, solid spore frog dung mold, cracked frog dung mold and the like. The above strains can be separated from soil and amphibian animal feces.
Cytotoxicity: at present, the incidence rate of cancer in China continues to increase, and the cancer becomes a public health challenge which cannot be ignored. According to public data statistics, the number of new tumor cases and death people in the nation in 2020 is listed as the first global, and the tumor prevention and treatment are indicated as the right of way. In the course of cancer treatment, a very important step is drug therapy, and the use of effective anticancer drugs can help the sick people to obtain longer survival time. The cytotoxicity test can evaluate the effectiveness, toxicity, drug resistance and the influence of the drug on the apoptosis, proliferation and the like of cancer cells, and has important value for the research and development of new anticancer drugs.
α -glucosidase inhibitory activity: type 2 diabetes (T2 DM) has become a serious public health problem and will affect the lives of 5 billion people worldwide by 2030. T2DM is characterized by disturbances in carbohydrate, lipid and protein metabolism, which are caused by a gradual decrease in insulin secretion from the pancreatic beta cells. Alpha-glucosidase was identified as a core target for the treatment of T2DM and carbohydrate diseases. Alpha-glucosides located on the surface membrane of the brush border of intestinal cells hydrolyze disaccharides or oligosaccharides to monosaccharides in the digestion of carbohydrates. Thus, inhibition of α -glucosidase activity may delay carbohydrate digestion, thereby alleviating T2DM. The α -glucosidase inhibitor competes for binding to α -glucosidase between the inhibitor and the di-or oligosaccharide, resulting in an inhibitory activity of α -glucosidase and a reduction in carbohydrate hydrolysis and glucose absorption.
Disclosure of Invention
The invention aims to provide cyclo-c-v-i-n-y-leu peptide with hepatotoxicity and alpha-glucosidase inhibitory activity and a preparation method thereof.
In order to find a novel natural and efficient substance with hepatotoxicity and alpha-glucosidase inhibitory activity, the inventor discovers that the cyclo-c-v-i-n-p-n-m-p-peptide extracted from a ranunculus japonicus has hepatotoxicity and alpha-glucosidase inhibitory activity through activity research on more than 100 strains of entomopathogenic fungi metabolites in China.
The structural formula of the cyclo-C-Val-isoleucin with hepatotoxicity and alpha-glucosidase inhibition activity is as follows:
the cyclo-tryptophan-alanine-valine-isoleucine-leucine peptide is a pentadecatomic cyclic pentapeptide which is formed by connecting tryptophan, alanine, valine, isoleucine and leucine through an amide bond;
the cyclo-c-v-i-leu peptide has hepatoma cytotoxicity and alpha-glucosidase inhibitory activity;
half Inhibitory Concentration (IC) against HepG2, a liver cancer cell 50 ) Comprises the following steps: 54.19 μ g/mL, half Inhibitory Concentration (IC) of alpha-glucosidase 50 ) Comprises the following steps: 63.64 μ g/mL.
The preparation operation steps of the cyclo-C-Val-Isoleu-leu peptide with hepatotoxicity and alpha-glucosidase inhibitory activity are as follows:
(1) Culture of bacterial species
Mixing Botrytis cinerea (A.ranunculata:)Basidiobolussp.) performing slant culture, secondary liquid seed culture, tertiary liquid seed culture and quaternary culture to obtain final liquid fermentation product or final solid fermentation product;
the frog dung mould (A), (B)Basidiobolussp.) is one of frogs of Rana chensinensis, rana chensinensis or Rana sidoides;
(2) Extraction and refining of effective components in the final fermentation
Extracting the effective components of the final fermented product with methanol or ethanol, and purifying by reverse phase chromatography to obtain white powdered cyclocro-C-Val-isoleu-leucin.
The preparation technical scheme is further defined as follows:
the specific operation of the step (1) is as follows:
(1.1) slant seed culture
Inoculating the frog dung mold strain to a potato agar (PDA) slant culture medium, and culturing at a constant temperature of 25-36 ℃ for 4-8 days to obtain a first-level strain;
(1.2) Secondary liquid seed culture
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 into a triangular flask with the volume of more than or equal to 100 ml, inoculating 1-3 slant strains into each triangular flask, and culturing for 3-7 d in a constant-temperature oscillation 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 composed of 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 (KCl), magnesium sulfate heptahydrate (MgSO) 4 .7H 2 0.3-2.0 g/L of O) and potassium dihydrogen phosphate (KH) 2 PO 4 ）0.3～2.0 g/L；
(1.3) three-stage liquid seed culture
Inoculating the second-level strain into a fermentation tank with the volume of more than or equal to 5L, wherein the liquid loading amount is 50-80% of the volume of the tank, the inoculation amount is 3-10%, and the third-level strain is obtained by carrying out constant-temperature aeration culture for 2-5 days under the conditions that the aeration amount is 1-2 (v/v) according to the volume ratio, the pressure is 0.1-0.2 MP and the temperature is 25-36 ℃;
the culture medium in the fermenter is synchronized with the liquid culture medium in step (1.2);
(1.4) four-stage culture
The fourth-stage culture is liquid culture, the third-stage strain is inoculated to a fermentation tank with the volume of more than or equal to 50L, the liquid loading amount is 50-80% of the volume of the tank, the inoculation amount is 3-10%, 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 amount is 1-2 (v/v) according to the volume ratio, the pressure is 0.1-0.2 MP and the temperature is 25-36 ℃; culture in fermenter liquid culture in step (1.2).
In the step (1.4), the fourth-stage culture is solid culture, the third-stage liquid strain is mixed into a solid culture medium, the inoculation amount is 3-10% of the solid material amount, and the solid final fermentation product is obtained after the constant-temperature culture at 25-36 ℃ for 5-15 days; the solid culture medium comprises 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 (KCl) and magnesium sulfate heptahydrate (MgSO) 4 .7H 2 0.3-2.0 g/L of O) and potassium dihydrogen phosphate (KH) 2 PO 4 ) 0.3-2.0 g/L, 15.0-30.0 g/L agar and water.
The specific operation of the step (2) is as follows:
(2.1) pretreatment of the final fermentation
Filtering the final fermentation product of the liquid through a filter membrane of 0.20-1.0 mu m or centrifuging the final fermentation product under the condition of 2000-12000rpm/min to obtain mycelium; freeze drying mycelium, crushing to obtain pretreated matter of 20-120 mesh;
or, drying the solid final fermentation product at 30-130 ℃ until the water content is less than or equal to 20%, and then crushing the solid final fermentation product into a pretreatment product with 20-120 meshes;
(2.2) extraction, separation and purification of effective components in the pretreated product
(2.2.1) extraction
Extracting the pretreated substance with methanol or ethanol; the material-liquid ratio is 1; centrifuging at a rotating speed of more than or equal to 2000rpm/min, or filtering with a 0.20-1.0 μm filter membrane; taking the filtrate or centrifugal supernatant, concentrating at the temperature of less than or equal to 100 ℃ to 1/2-1/10 of the original volume to obtain concentrated dealcoholized liquid, simultaneously recovering methanol or ethanol, and using the concentrated dealcoholized liquid for further refining by chromatography;
(2.2.2) separation and purification
Purifying by adopting a reverse phase chromatography method, wherein the stationary phase is bonding phase filler, the bonding phase filler is reverse carbon eighteen filler (RPC 18), and the particle diameter is 3-50 mu m; methanol and water according to the weight ratio of 20-70: gradient elution is carried out in a proportion of 80 to 30, and chromatographic fractions with the molecular weight of 583 on a mass spectrum detector are collected; concentrating the collected substance to viscous state at a temperature of less than or equal to 100 ℃, and drying at a temperature of less than 170 ℃; the cyclophrome-c-v-iso-leu peptide was obtained as a white powder.
The analytical study of the present invention is illustrated below:
1. screening research discovers a frog dung mouldBasidiobolussp.) the mycelium methanol extract has strong hepatotoxicity and alpha-glucosidase inhibitory activity:
(1) Cytotoxicity determination of methanol extract on hepatoma cell HepG 2: preparing DMSO solution with concentration of 1, 10, 25, 50, 100 μ g/ml, performing cytotoxicity inhibition test with podophyllotoxin as positive control by CCK-8 method, measuring absorbance at 450 nm, and calculating to obtain half Inhibition Concentration (IC) of extract on liver cancer cell HepG2 50 ) Comprises the following steps: 210.32. Mu.g/mL;
(2) Determination of alpha-glucosidase inhibitory activity of methanol extract: preparing 10, 100, 250, 500 and 1000 mu g/ml methanol solution of the extract sample, measuring the activity of the extract on alpha-glucosidase at 405nm, and calculating to obtain the semi-Inhibitory Concentration (IC) of the extract on the alpha-glucosidase 50 ) Comprises the following steps: 1352.89 μ g/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) The cytotoxicity of the cyclo-C-Val-Ile-Leucin on liver cancer cell HepG2 is determined as follows: preparing DMSO solution with concentration of 1, 10, 25, 50, 100 μ g/ml, performing cytotoxicity inhibition test with podophyllotoxin as positive control by CCK-8 method, measuring absorbance at 450 nm, and calculating to obtain half Inhibition Concentration (IC) of extract on liver cancer cell HepG2 50 ) Comprises the following steps: 54.19 Mu g/mL;
(2) Determination of α -glucosidase inhibitory activity of cyclo-c-v-i-leu peptide: preparing 10, 100, 250, 500, 1000 μ g/ml methanol solution of the extract sample, measuring alpha-glucosidase activity at 405nm, and calculating to obtain semi-Inhibitory Concentration (IC) of the extract on alpha-glucosidase 50 ) Comprises the following steps: 63.64 μ g/mL.
3. Chemical Structure characterization of active Compounds
High resolution LC-MS analysis showed that the purified active compound cation 583.3611 (M + H) + )、605.3428(M+Na + ) Calculated molecular formula C 31 H 46 N 6 O 5 。
Nmr data are shown in the following table:
| unit | carbon | δ C ， type | δ H ， (J in Hz) | unit | carbon | δ C ， type | δ H ， (J in Hz) |
| Trp | 1 | 171.5， C | 17 | 29.5， CH | 1.86， m | ||
| 2 | 56.4， CH | 4.32， dt (7.4， 7.3) | 18 | 19.5， CH 3 | 0.82， d (6.8) | ||
| 3a | 27.7， CH2 | 2.93， dd (14.6， 7.4) | 19 | 19.6， CH 3 | 0.82， d (6.8) | ||
| 3b | 3.00， dd (14.6， 7.3) | 16-NH | 8.26， d (7.8) | ||||
| 4 | 110.4， C | Ile | 20 | 171.3， C | |||
| 5 | 123.9， CH | 7.08， d (1.9) | 21 | 57.0， CH | 4.11， m | ||
| NH | 10.80， d (1.8) | 22 | 37.3， CH | 1.68， m | |||
| 6 | 136.6， C | 23a | 25.3， CH 2 | 0.97， m | |||
| 7 | 111.8， CH | 7.28， d (8.0) | 23b | 1.03， m | |||
| 8 | 121.4， CH | 7.00， t (7.5) | 24 | 11.9， CH 3 | 0.77， d (6.8) | ||
| 9 | 118.8， CH | 6.93， t (7.5) | 25 | 15.4， CH 3 | 0.80， overlap | ||
| 10 | 118.8， CH | 7.50， d (7.9) | 21-NH | 8.05， d (7.8) | |||
| 11 | 127.7， C | Leu | 26 | 172.4， C | |||
| 2-NH | 8.35， br s | 27 | 55.3， CH | 3.92， dt (8.9， 8.0) | |||
| Ala | 12 | 171.8， C | 28a | 40.9， CH 2 | 1.39， m | ||
| 13 | 49.0， CH | 4.14， m | 28b | 1.53， m | |||
| 14 | 17.7， CH 3 | 1.09， d (6.8) | 29 | 24.9， CH | 1.21， m | ||
| 13-NH | 7.56， d (6.9) | 30 | 22.3， CH 3 | 0.69， d (6.5) | |||
| Val | 15 | 171.3， C | 31 | 23.1， CH 3 | 0.75， d (6.6) | ||
| 16 | 61.5， CH | 3.69， t (7.8) | 27-NH | 8.62， br s |
the structure of the separated and purified active compound is the cyclochrolo-C-Val-isoleucin-leucin which is obtained by comprehensive liquid chromatography-mass spectrometry analysis and nuclear magnetic resonance analysis and is shown as the chemical structural formula.
The beneficial technical effects of the invention are embodied in the following aspects:
1. the cyclo-C-Val-isoleucin-leucin prepared by the method has hepatotoxicity and alpha-glucosidase inhibitory activity, and develops a new application field of the frog dung mould. The invention discovers that the frogspawn has the function of metabolizing the active substances for the first time. On the basis of the invention, related genes can be expected to be further cloned to construct high-yield strains.
2. The cyclo-C-V-I-L-leucine peptide is a novel skeleton and can be used as a medicine lead compound for derivatization modification to create a compound with more and stronger activity.
3. The cyclo-C-Val-isoleucin can be used as a potential drug for treating liver cancer and weight loss.
4. The invention adopts the microbial fermentation production, is not influenced by environment and resources, 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 present invention will be further described with reference to the following examples.
Example 1:
the preparation operation steps of the cyclo-C-Val-Isoleu-leu peptide with hepatotoxicity and alpha-glucosidase inhibitory activity are as follows:
1.1 Rana Nigromaculata Strain selection
The Rana chensinensis mould is Rana chensinensis mould;
1.2 Strain culture
The culture method is liquid culture and comprises the following specific steps:
1.2.1 slant seed culture
Inoculating the preserved Rana Nigromaculata strain to potato agar (PDA) slant culture medium, and culturing at 25 deg.C for 4 days to obtain first-class strain;
1.2.2 Secondary liquid seed culture
Inoculating the first-level strain to a liquid shake flask culture medium for culture;
liquid shake flask culture medium: 25.0g/L glucose, 10.0g/L malt extract, 2.0 g/L peptone, 1.0g/L yeast extract powder, 0.3g/L potassium chloride (KCl), magnesium sulfate heptahydrate (MgSO) 4 .7H 2 O) 0.3g/L, potassium dihydrogen phosphate (KH) 2 PO 4 ）0.3g/L；
Adding a liquid culture medium with the volume of 30% of that of a triangular flask into a 100 ml triangular flask, inoculating 1 slant strain into each triangular flask, placing the triangular flasks in a constant-temperature oscillation incubator after inoculation, and culturing at the temperature of 25 ℃ and the rotating speed of 150 rpm for 3d to obtain a secondary strain;
1.2.3 three-stage liquid seed culture
Inoculating the second-level strain into a 5L fermentation tank, wherein the liquid loading amount is 50% of the volume of the tank, the inoculation amount is 3%, the ventilation amount is 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.
Quaternary culture
The four-stage culture adopts liquid culture: inoculating the third-level strain into a 50L fermentation tank, wherein the liquid loading amount is 50% of the tank volume, the inoculation amount is 3%, the ventilation amount is 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 liquid; the culture medium in the fermentation tank is the same as the liquid shake flask culture medium;
1.3 extraction and purification of active ingredients in the final fermented product
1.3.1 pretreatment of the final fermentate
Filtering the final fermentation product of the liquid through a 0.20 mu m filter membrane to obtain mycelium; the mycelium was freeze-dried and ground to 20 mesh for use.
Extraction of
Extracting the above dried mycelium with methanol; the material-liquid ratio is 1; centrifuging at 2000rpm/min, concentrating the supernatant at 100 deg.C to 1/2 of the original volume to obtain concentrated dealcoholized solution, recovering methanol, and refining by chromatography.
Separating and purifying
Purifying by reverse phase chromatography. The stationary phase is bonding phase filler, the bonding phase filler is reverse carbon eighteen filler (RPC 18), the particle diameter is 3 mu m, and 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 added according to the proportion of 20-70: gradient elution is carried out in a proportion of 80-30, and a target peak with the molecular weight of 583 on a mass spectrum detector is collected; concentrating the collected substance to be viscous at the temperature of below 100 ℃, and drying at the temperature of 170 ℃; a white powder of cyclo-c-v-iso-leu peptide was obtained.
The structural identification result shows that the structural formula of the cyclo-C-Val-isoleucin is as follows:
the activity measurement result shows that the half-Inhibitory Concentration (IC) of the cyclo-C-Val-isoleucin on the hepatoma cell HepG2 50 ) Comprises the following steps: 54.19 μ g/mL, half Inhibitory Concentration (IC) of alpha-glucosidase 50 ) Comprises the following steps: 63.64 μ g/mL.
Example 2
Has hepatotoxicity and alpha-glucosidase inhibitory activity the operation steps for preparing the cyclo-C-Val-isoleucin are as follows:
2.1 selection of Botrytis frog-dung mould Strain
The Rana chensinensis David dung mold is Botrytis cinerea;
2.2 Strain culture
The culture method is liquid culture and comprises the following specific steps:
2.2.1 slant seed culture
Inoculating the preserved Rana chensinensis Mucor strain to potato agar (PDA) slant culture medium, and culturing at 36 deg.C for 8 days to obtain first-class strain;
2.2.2 Secondary liquid seed culture
Inoculating the first-stage strain to a liquid shake flask culture medium for culture;
liquid shake flask culture medium: 50.0 g/L glucose, 30.0 g/L malt extract, 6.0 g/L peptone, 4.0 g/L yeast extract powder, 0.3g/L potassium chloride (KCl), magnesium sulfate heptahydrate (MgSO) 4 .7H 2 O) 0.3g/L, potassium dihydrogen phosphate (KH) 2 PO 4 ）2.0 g/L。
Adding liquid culture medium 50% of the volume of the triangular flask into 1000 ml of triangular flasks, inoculating 3 slant strains into each triangular flask, placing in a constant temperature oscillation incubator after inoculation, culturing at 36 ℃ and 250 rpm for 7d to obtain secondary strains.
Three stage liquid seed culture
Inoculating the second-level strain into a 50L fermentation tank, wherein the liquid loading amount is 80% of the volume of the tank, the inoculation amount is 10%, and the aeration amount is 1 (v/v) in volume ratio, 2 (v/v), the pressure is 0.2MP, and the constant-temperature aeration culture is carried out for 5 days at 36 ℃ to obtain a third-level strain.
The culture medium in the fermentation tank is the same as the liquid shake-flask culture medium.
Four stage culture
The fourth-stage culture is liquid culture: inoculating the third-level strain into a fermentation tank with volume of 500L or more, wherein the liquid loading amount is 80% of the tank volume, the inoculation amount is 10%, and the aeration amount is 1 (v/v) in volume ratio, 0.2MP and 36 ℃ for constant-temperature aeration culture for 15 days to obtain a final liquid fermentation product; the culture medium in the fermentation tank is the same as the liquid shake flask culture medium.
Extraction and refining of effective components in the final fermentation
2.3.1 pretreatment of the final fermentate
Filtering the final fermented product with 1.0 μm filter membrane to obtain mycelium; the mycelium was freeze-dried and ground to 120 mesh for use.
Extraction of
Extracting the dried mycelium with ethanol at a material-liquid ratio of 1; centrifuging at 12000rpm/min, concentrating the supernatant at 40 deg.C to 1/10 of the original volume to obtain concentrated dealcoholized liquid, recovering ethanol, and refining with chromatography.
Separating and purifying
Purifying by reverse phase chromatography. The stationary phase is bonding phase filler, the bonding phase filler is reverse carbon eighteen filler (RPC 18), the particle diameter is 50 μm, and the chromatographic column diameter and the flow rate of the mobile phase can be determined according to the production scale; methanol and water are added according to the weight ratio of 20-70: gradient elution is carried out in a proportion of 80-30, and a target peak with the molecular weight of 583 on a mass spectrum detector is collected; concentrating the collected substance to be viscous at the temperature of below 30 ℃, and drying at the temperature of-30 ℃; the cyclophrome-c-v-iso-leu peptide was obtained as a white powder.
The result of the structure identification shows that, the structural formula of the cyclo-c-v-i-n-leucin is as follows:
the activity measurement result shows that the half-Inhibitory Concentration (IC) of the cyclo-C-Val-Illustration-Leucin on the hepatoma cell HepG2 50 ) Comprises the following steps: 54.19 μ g/mL, half Inhibitory Concentration (IC) of alpha-glucosidase 50 ) Comprises the following steps: 63.64 mu.g/mL.
Example 3:
has hepatotoxicity and alpha-glucosidase inhibitory activity the operation steps for preparing the cyclo-C-Val-isoleucin are as follows:
3.1 frog-dung mould Strain selection
The selected strain is ranunculus glaucocalyx;
3.2 Strain culture
The culture method is liquid-solid mixed culture and comprises the following specific steps:
3.2.1 slant seed culture
Inoculating the preserved Rana Nigromaculata strain to potato agar (PDA) slant culture medium, and culturing at 30 deg.C for 6 days to obtain first-stage strain;
3.2.2 Secondary liquid seed culture
Inoculating the first-level strain to a liquid shake flask culture medium for culture;
liquid shake flask culture medium: 35.0 g/L glucose, 20.0 g/L malt extract, 4.0 g/L peptone, 2.0 g/L yeast extract powder, 0.3g/L potassium chloride (KCl), magnesium sulfate heptahydrate (MgSO) 4 .7H 2 O) 0.3g/L, potassium dihydrogen phosphate (KH) 2 PO 4 ）1.0 g/L。
Adding liquid culture medium 40% of the volume of the triangular flask into 500 ml of triangular flasks, inoculating 2 slant strains into each triangular flask, placing the triangular flasks in a constant-temperature oscillation incubator after inoculation, and culturing for 5d at the temperature of 30 ℃ and the rotating speed of 200 rpm to obtain secondary strains.
Three stage liquid seed culture
Inoculating the second-level strain into a 50L fermentation tank, wherein the liquid loading amount is 60% of the volume of the tank, the inoculation amount is 6%, the ventilation amount is 1.5 (v/v) according to the volume ratio of 1, the pressure is 0.15MP, and the constant-temperature ventilation culture is carried out for 3 days at 30 ℃ to obtain a third-level strain.
The culture medium in the fermentation tank is the same as the liquid shake flask culture medium.
Four stage culture
The fourth-stage culture is solid culture: mixing the third-stage liquid strain with solid culture medium, inoculating 3% of the solid material, and performing constant temperature aeration culture at 25 deg.C for 10 days to obtain solid final fermented product; the solid culture medium is as follows: 25.0g/L glucose, 10.0g/L malt extract, 2.0 g/L peptone, 1.0g/L yeast extract powder, 0.3g/L potassium chloride (KCl), 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.
Extraction and refining of effective components in the final fermentation
3.3.1 pretreatment of the final fermentate
The solid final fermentation was dried at 30 ℃ to a moisture content of 20% and then ground to 20 mesh for use.
Extraction of
Extracting the dried and pulverized final fermented product with ethanol; the material-liquid ratio is 1; filtering with 0.20 μm filter membrane after extraction; concentrating the filtrate at 30 deg.C to 1/2 of the original volume to obtain concentrated dealcoholized liquid, recovering ethanol, and refining by chromatography;
3.3.3 Separating and purifying
Purifying by reverse phase chromatography. The stationary phase is bonding phase filler, the bonding phase filler is reverse carbon eighteen filler (RPC 18), the particle diameter is 25 μm, and the chromatographic column diameter and the flow rate of the mobile phase can be determined according to the production scale; methanol and water are added according to the weight ratio of 20-70: gradient elution is carried out in a proportion of 80-30, and a target peak with the molecular weight of 583 on a mass spectrum detector is collected; concentrating the collected substance to be viscous at the temperature of below 40 ℃, and drying at the temperature of 10 ℃; the cyclophrome-c-v-iso-leu peptide was obtained as a white powder.
The result of the structure identification shows that, the structural formula of cyclo-c-v-i-leu peptide is as follows:
the activity measurement result shows that the half-Inhibitory Concentration (IC) of the cyclo-C-Val-isoleucin on the liver cancer cell HepG2 50 ) Comprises the following steps: 54.19 μ g/mL, half Inhibitory Concentration (IC) of alpha-glucosidase 50 ) Comprises the following steps: 63.64 μ g/mL.
Example 4:
has hepatotoxicity and alpha-glucosidase inhibitory activity the operation steps for preparing the cyclo-C-Val-isoleucin are as follows:
4.1 Rana Nigromaculata strains selection
The frog fecal mould is frog raw frog fecal mould;
4.2 Strain culture
The culture method is liquid-solid mixed culture and comprises the following specific steps:
4.2.1 slant seed culture
Inoculating the preserved Rana Nigromaculata strain to potato agar (PDA) slant culture medium, and culturing at 29 deg.C for 5d to obtain first-stage strain;
4.2.2 Secondary liquid seed culture
Inoculating the first-stage strain to a liquid shake flask culture medium for culture;
liquid shake flask culture medium: glucose 40.0 g/L, malt extract 25.0g/L, peptone 3.0 g/L, yeast extract powder 3.0 g/L, potassium chloride (KCl) 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。
Adding liquid culture medium 40% of the volume of the triangular flask into 500 ml of triangular flasks, inoculating 1 slant strain into each triangular flask, placing the triangular flasks in a constant-temperature oscillation incubator after inoculation, and culturing for 4d at the temperature of 30 ℃ and the rotating speed of 180 rpm to obtain a secondary strain.
Three stage liquid seed culture
Inoculating the second-level strain into a 30L fermentation tank, wherein the liquid loading amount is 65% of the volume of the tank, the inoculation amount is 6%, and the aeration amount is 1.2 (v/v) according to the volume ratio of 1, the pressure is 0.12MP, and the constant-temperature aeration culture is carried out for 3 days at the temperature of 32 ℃ to obtain a third-level strain.
The culture medium in the fermentation tank is the same as the liquid shake flask culture medium.
Four stage culture
The fourth-stage culture adopts solid culture: mixing the third-stage liquid strain with solid culture medium, inoculating 10% of the solid material, and performing constant temperature aeration culture at 36 deg.C for 8 days to obtain solid final fermented product;
the solid culture medium is: 50.0 g/L glucose, 30.0 g/L malt extract, 6.0 g/L peptone, 4.0 g/L yeast extract powder, 0.3g/L potassium chloride (KCl), magnesium sulfate heptahydrate (MgSO) 4 .7H 2 O) 0.3g/L, potassium dihydrogen phosphate (KH) 2 PO 4 ) 2.0 g/L of agar and 30.0 g/L of agar.
Extraction and refining of effective components in the final fermentation
4.3.1 pretreatment of the final fermentate
The solid final fermentation product was dried at 130 ℃ to a water content of 10% and then pulverized to 120 mesh for use.
Extraction of
Extracting the dried and crushed final fermented product with methanol at a feed-liquid ratio of 1 (W: V) and ultrasonic power of 100KHz for 200 min; filtering with 1.0 μm filter membrane after extraction; concentrating the filtrate at 40 deg.C to 1/10 of the original volume to obtain concentrated dealcoholized liquid, recovering methanol, and further refining by chromatography;
4.3.3 Separating and purifying
Purifying by reverse phase chromatography. The stationary phase is bonding phase filler, the bonding phase filler is reverse carbon eighteen filler (RPC 18), the particle diameter is 10 μm, and the chromatographic column diameter and the flow rate of the mobile phase can be determined according to the production scale; methanol and water are added according to the weight ratio of 20-70: gradient elution is carried out in a proportion of 80-30, and a target peak with the molecular weight of 583 on a mass spectrum detector is collected; concentrating the collected substance to be viscous at the temperature of below 100 ℃, and drying at the temperature of 170 ℃; the cyclophrome-c-v-iso-leu peptide was obtained as a white powder.
The result of the structure identification shows that, the structural formula of cyclo-c-v-i-leu peptide is as follows:
the activity measurement result shows that the half-Inhibitory Concentration (IC) of the cyclo-C-Val-isoleucin on the liver cancer cell HepG2 50 ) Comprises the following steps: 54.19 μ g/mL, half Inhibitory Concentration (IC) of alpha-glucosidase 50 ) Comprises the following steps: 63.64 μ g/mL.
Claims (5)
1. A cyclo-chromo-C-valine-isoleucyl leucine peptide with hepatotoxicity and alpha-glucosidase inhibition activity, characterized in that: the structural formula of the cyclo-chromo-C-Val-isoleucin is as follows:
the cyclo-tryptophan-alanine-valine-isoleucine-leucine peptide is a pentadecatomic cyclic pentapeptide which is formed by connecting tryptophan, alanine, valine, isoleucine and leucine through an amide bond;
the cyclo-c-v-i-leu peptide has hepatoma cytotoxicity and alpha-glucosidase inhibitory activity; half Inhibitory Concentration (IC) against HepG2, a liver cancer cell 50 ) Comprises the following steps: 54.19 μ g/mL, half Inhibitory Concentration (IC) of alpha-glucosidase 50 ) Comprises the following steps: 63.64 μ g/mL.
2. The method for preparing cyclo-chromo-c-v-i-leu peptide with hepatotoxicity and α -glucosidase inhibitory activity of claim 1, characterized by the following steps:
(1) Culture of bacterial species
Mixing all the wood frogs of Rana NigromaculataBasidiobolussp.) performing slant culture, secondary liquid seed culture, tertiary liquid seed culture and quaternary culture to obtain final liquid fermentation product or final solid fermentation product;
the frog dung mould (A), (B)Basidiobolussp.) is one of frogs of Rana chensinensis, rana chensinensis or Rana sidoides;
(2) Extraction and refining of effective components in the final fermentation
Extracting the effective components of the final fermented product with methanol or ethanol, and purifying by reverse phase chromatography to obtain white powdered cyclocro-C-Val-isoleu-leucin.
3. The method according to claim 2, wherein the step (1) is specifically performed by:
(1.1) slant seed culture
Inoculating the frog dung mold strain to a potato agar (PDA) slant culture medium, and culturing at a constant temperature of 25-36 ℃ for 4-8 days to obtain a first-level strain;
(1.2) Secondary liquid seed culture
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 into a triangular flask with the volume of more than or equal to 100 ml, inoculating 1-3 slant strains into each triangular flask, and culturing for 3-7 d in a constant-temperature oscillation 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 composed of 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 (KCl), magnesium sulfate heptahydrate (MgSO) 4 .7H 2 0.3-2.0 g/L of O) and potassium dihydrogen phosphate (KH) 2 PO 4 ）0.3～2.0 g/L；
(1.3) three-stage liquid seed culture
Inoculating the second-level strain into a fermentation tank with the volume of more than or equal to 5L, wherein the liquid loading amount is 50-80% of the volume of the tank, the inoculation amount is 3-10%, and the third-level strain is obtained by carrying out constant-temperature aeration culture for 2-5 days under the conditions that the aeration amount is 1-2 (v/v) according to the volume ratio, the pressure is 0.1-0.2 MP and the temperature is 25-36 ℃;
the culture medium in the fermenter is synchronized with the liquid culture medium in step (1.2);
(1.4) four-stage culture
The fourth-stage culture is liquid culture, the third-stage strain is inoculated to a fermentation tank with the volume of more than or equal to 50L, the liquid loading amount is 50-80% of the volume of the tank, the inoculation amount is 3-10%, 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 amount is 1-2 (v/v) according to the volume ratio, the pressure is 0.1-0.2 MP and the temperature is 25-36 ℃; culture in fermenter liquid culture in step (1.2).
4. The method according to claim 3, wherein: in the step (1.4), the fourth-stage culture is solid culture, the third-stage liquid strain is mixed into a solid culture medium, the inoculation amount is 3-10% of the solid material amount, and the solid final fermentation product is obtained after the constant-temperature culture at 25-36 ℃ for 5-15 days; the solid culture medium comprises 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 (KCl) and magnesium sulfate heptahydrate (MgSO) 4 .7H 2 0.3-2.0 g/L of O) and potassium dihydrogen phosphate (KH) 2 PO 4 ) 0.3-2.0 g/L, 15.0-30.0 g/L agar and water.
5. The method according to claim 2, wherein the step (2) is specifically performed by:
(2.1) pretreatment of the final fermentation
Filtering the final fermented product with 0.20-1.0 μm filter membrane or centrifuging at 2000-12000rpm/min to obtain mycelium; freeze-drying the mycelium, and crushing to obtain a pretreated substance which is sieved by a sieve of 20-120 meshes;
or, drying the solid final fermentation product at 30-130 ℃ until the water content is less than or equal to 20%, and then crushing the solid final fermentation product into a pretreatment product with 20-120 meshes;
(2.2) extraction, separation and purification of effective components in the pretreated product
(2.2.1) extraction
Extracting the pretreated substance with methanol or ethanol; the material-liquid ratio is 1; centrifuging at a rotating speed of more than or equal to 2000rpm/min, or filtering with a 0.20-1.0 μm filter membrane; taking the filtrate or supernatant after centrifugation, concentrating at the temperature of less than or equal to 100 ℃ to 1/2-1/10 of the original volume to obtain concentrated dealcoholized liquid, simultaneously recovering methanol or ethanol, and using the concentrated dealcoholized liquid for further chromatographic refining;
(2.2.2) separation and purification
Purifying by adopting a reverse phase chromatography method, wherein the stationary phase is bonding phase filler, the bonding phase filler is reverse carbon eighteen filler (RPC 18), and the particle diameter is 3-50 mu m; methanol and water are added according to the weight ratio of 20-70: gradient elution is carried out in a proportion of 80 to 30, and chromatographic fractions with the molecular weight of 583 on a mass spectrum detector are collected; concentrating the collected substance to viscous state at a temperature of less than or equal to 100 ℃, and drying at a temperature of less than 170 ℃; the cyclophrome-c-v-iso-leu peptide was obtained as a white powder.
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