CN115651067B - Cyclic color-propyl-valyl-isoleucyl-leucinide with hepatoma cytotoxicity and alpha-glucosidase inhibitory activity and preparation method thereof - Google Patents
Cyclic color-propyl-valyl-isoleucyl-leucinide with hepatoma cytotoxicity and alpha-glucosidase inhibitory activity and preparation method thereof Download PDFInfo
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Abstract
The invention relates to a cyclophilin-propyl-valyl-isoleucyl-leucinide (Trp-Ala-Val-Ile-Leu cyclopeptide) with hepatoma cytotoxicity and alpha-glucosidase inhibitory activity and a preparation method thereof, belonging to the technical field of extraction and preparation of biological products. The cyclic-propyl-valyl-isoleucyl-leucinide is fifteen-membered cyclic pentapeptide formed by connecting tryptophan, alanine, valine, isoleucine and leucine through an amide bond. This 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 ring color-propyl-valyl-isoleucyl-leucinyl peptide is white powder, and the half inhibition concentration (IC 50) of the pure product on liver cancer cells HepG2 is as follows: 54.19 μg/mL, half inhibitory concentration of α -glucosidase (IC 50) was: 63.64. Mu.g/mL. The compound can be used as potential medicine for treating liver cancer and slimming medicine.
Description
Technical Field
The invention belongs to the technical field of extraction and preparation of biological products, and relates to extraction and purification of cyclophosphamide-propyl-valyl-isoleucyl-leucinide (Trp-Ala-Val-Ile-Leu cyclopeptide) with hepatoma cytotoxicity and alpha-glucosidase inhibitory activity.
Background
The cyclophilin-propyl-valyl-isoleucyl-leucinide is a bioactive substance with hepatoma cytotoxicity and alpha-glucosidase inhibitory activity, which is separated from a culture of frog feces mould (Basidiokutsp.).
The Rana Nigromaculata (Basidiokusp.) is fungus of Rana Nigromaculata of Rana of 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 separated from soil and amphibian feces.
Cytotoxicity: at present, the incidence of cancer in China continues to increase, and the cancer has become a non-negligible public health challenge. In the cancer treatment process, a quite important link is drug treatment, and effective anticancer drugs are used, so that patients can be helped to obtain longer survival time. The cytotoxicity experiment can evaluate the effects of the effectiveness, toxicity, drug resistance and the effects of the drugs on cancer cell apoptosis, proliferation and the like, and has important value for the research and development of new anticancer drugs.
Alpha-glucosidase inhibitory Activity: type 2 diabetes (T2 DM) has become a serious public health problem, affecting the lives of 5 million people worldwide by 2030. T2DM is characterized by a disturbance in carbohydrate, lipid and protein metabolism, which is caused by the gradual decrease in insulin secretion by pancreatic beta cells. Alpha-glucosidase was identified as a core target for the treatment of T2DM and carbohydrate diseases. Alpha-glucosides located in the brush border surface membrane of intestinal cells hydrolyze di-or oligosaccharides to monosaccharides in carbohydrate digestion. Thus, inhibition of α -glucosidase activity can delay carbohydrate digestion, thereby alleviating T2DM. The alpha-glucosidase inhibitors compete between the inhibitor and di-or oligosaccharides for binding to alpha-glucosidase, resulting in inhibition activity of alpha-glucosidase and a reduction in carbohydrate hydrolysis and glucose absorption.
Disclosure of Invention
The invention aims to provide a cyclophosphamide-propyl-valyl-isoleucyl peptide with hepatoma cytotoxicity and alpha-glucosidase inhibitory activity and a preparation method thereof.
In order to find novel natural and efficient substances with hepatoma cytotoxicity and alpha-glucosidase inhibitory activity, the inventor finds out that the cyclophosphamide-propyl-valyl-isoleucin extracted from the frog-in-the-middle-um has the effects of hepatoma cytotoxicity and alpha-glucosidase inhibitory activity through carrying out activity research on more than 100 insect pathogen fungi metabolites in China.
The structural formula of the cyclophospha-propan-valyl-isoleucyl-leucinide with hepatoma cytotoxicity and alpha-glucosidase inhibitory activity is as follows:
The cyclic-propyl-valyl-isoleucyl-leucinide is fifteen-membered cyclic pentapeptide formed by connecting tryptophan, alanine, valine, isoleucine and leucine through an amide bond;
the cyclophosphamide-propyl-valyl-isoleucyl-leucinide has liver cancer cytotoxicity and alpha-glucosidase inhibitory activity;
the half inhibitory concentration (IC 50) on hepatoma cell HepG2 was: 54.19 The half inhibitory concentration (IC 50) of α -glucosidase in μg/mL is: 63.64 μg/mL.
The preparation method of the cyclophosphamide-propyl-valyl-isoleucyl-leucinide with hepatoma cytotoxicity and alpha-glucosidase inhibitory activity comprises the following steps:
(1) Strain culture
Culturing Rana Nigromaculata (Basidiokusp.) by slant strain, secondary liquid seed culture, tertiary liquid seed culture and quaternary culture to obtain final fermentation product of liquid or solid;
the frog-excrement mould (Basidiokulsp) is one of frog-growth frog-excrement mould, solid-spore frog-excrement mould or schizosporium-excrement mould;
(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-propyl-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 seed culture
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) two-stage 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 with the volume of more than or equal to 100ml into the triangular flask, inoculating 1-3 inclined plane strains into each triangular flask, and culturing for 3-7 d in a constant-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 25.0-50.0 g/L glucose, 10.0-30.0 g/L malt extract, 2.0-6.0 g/L peptone g/L yeast extract powder 1.0-4.0 g/L potassium chloride (KCl) 0.3-2.0 g/L, magnesium sulfate heptahydrate (MgSO 4.7H2 O) 0.3-2.0 g/L, potassium dihydrogen phosphate (KH 2PO4) 0.3-2.0 g/L;
(1.3) three-stage liquid seed 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, 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 (KCl), 0.3-2.0 g/L of magnesium sulfate heptahydrate (MgSO 4.7H2 O), 0.3-2.0 g/L of potassium dihydrogen phosphate (KH 2PO4), 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 filter membrane with the thickness of 0.20-1.0 μm or centrifuging at the speed of 2000-12000rpm/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 more than or equal to 2000rpm/min, or filtering with a 0.20-1.0 mu m 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 mu m; methanol and water are used according to the proportion of 20 to 70: gradient elution is carried out according to the proportion of 80-30, and chromatographic fractions with the molecular weight of 583 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-propyl-valyl-isoleucyl-leucinide was obtained as a white powder.
The analytical study of the present invention is described as follows:
1. Screening researches show that the frog-faecalis (Basidiokutssp.) mycelium methanol extract has strong hepatoma cytotoxicity and alpha-glucosidase inhibition activity:
(1) Cytotoxicity assay of methanol extract on hepatoma cells HepG 2: preparing DMSO solution with the concentration of 1,10, 25, 50 and 100 mug/ml, taking podophyllotoxin as a positive control, performing cytotoxicity inhibition test by using a CCK-8 method, measuring absorbance at 450 nm, and calculating to obtain the extract with the semi-inhibition concentration (IC 50) of HepG2 liver cancer cell: 210.32 μg/mL;
(2) Determination of alpha-glucosidase inhibitory Activity of methanol extract: the concentration of the extract sample is 10, 100, 250, 500, 1000 mug/ml methanol solution, the activity of the extract on alpha-glucosidase is measured at 405nm, and the semi-inhibition concentration (IC 50) of the extract on the alpha-glucosidase is calculated as follows: 1352.89 μg/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) Cytotoxicity assay of cyclophilin-pro-valiso-leu-leucins on hepatoma cells HepG 2: preparing DMSO solution with the concentration of 1, 10, 25, 50 and 100 mug/ml, taking podophyllotoxin as a positive control, performing cytotoxicity inhibition test by using a CCK-8 method, measuring absorbance at 450 nm, and calculating to obtain the extract with the semi-inhibition concentration (IC 50) of HepG2 liver cancer cell: 54.19 μg/mL;
(2) Determination of alpha-glucosidase inhibitory activity of cyclophosphamide-propan-valyl-isoleucyl-leucinyl: the concentration of the extract sample is 10, 100, 250, 500, 1000 mug/ml methanol solution, the activity of the extract on alpha-glucosidase is measured at 405nm, and the semi-inhibition concentration (IC 50) of the extract on the alpha-glucosidase is calculated as follows: 63.64 μg/mL.
3. Chemical structure identification of active compounds
High resolution liquid chromatography mass spectrometry analysis showed purified active compound positive ion 583.3611 (m+h +)、605.3428(M+Na+), calculated molecular formula C 31H46N6O5.
The nmr analysis data are shown in the following table:
| unit | carbon | δC，type | δH，(Jin Hz) | unit | carbon | δC，type | δH，(Jin 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， CH3 | 0.82， d (6.8) | ||
| 3a | 27.7， CH2 | 2.93， dd (14.6， 7.4) | 19 | 19.6， CH3 | 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， CH2 | 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， CH3 | 0.77， d (6.8) | ||
| 9 | 118.8， CH | 6.93， t (7.5) | 25 | 15.4， CH3 | 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， CH2 | 1.39， m | ||
| 13 | 49.0， CH | 4.14， m | 28b | 1.53， m | |||
| 14 | 17.7， CH3 | 1.09， d (6.8) | 29 | 24.9， CH | 1.21， m | ||
| 13-NH | 7.56， d (6.9) | 30 | 22.3， CH3 | 0.69， d (6.5) | |||
| Val | 15 | 171.3， C | 31 | 23.1， CH3 | 0.75， d (6.6) | ||
| 16 | 61.5， CH | 3.69， t (7.8) | 27-NH | 8.62， br s |
the active compound structure obtained by separation and purification is ring color-propyl-valyl-isoleucyl-leucinyl peptide, which is obtained by comprehensive liquid chromatography-mass spectrometry analysis and nuclear magnetic resonance analysis, and the chemical structural formula is shown.
The beneficial technical effects of the invention are shown in the following aspects:
1. The cyclophosphamide-propyl-valyl-isoleucyl-leucinide prepared by the method has liver cancer cytotoxicity and alpha-glucosidase inhibition activity, and opens up a new application field of frog-feces mould. 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-propyl-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-propyl-valyl-isoleucyl-leucinide can be used as a potential drug for treating liver cancer and slimming drugs.
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 cyclophosphamide-propyl-valyl-isoleucyl-leucinide with hepatoma cytotoxicity and alpha-glucosidase inhibitory activity comprises the following steps:
1.1 selection of frog feces mould seed
The used frog-feces mold is frog-raw frog-feces mold;
1.2 Strain culture
The culture method is liquid culture, and comprises the following specific procedures:
1.2.1 slant seed culture
Inoculating the preserved frog-faecal fungus strain into potato agar (PDA) slant culture medium, and culturing at 25deg.C for 4d to obtain first-class strain;
1.2.2 two-stage liquid seed culture
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, 2.0 g/L peptone, 1.0g/L yeast extract, 0.3g/L potassium chloride (KCl), 0.3g/L magnesium sulfate heptahydrate (MgSO 4.7H2 O), 0.3g/L potassium dihydrogen phosphate (KH 2PO4);
Adding 30% of the volume of a triangular flask into a triangular flask of 100 ml, inoculating 1 inclined plane strain to each triangular flask, placing the triangular flask into a constant-temperature shaking incubator at 25 ℃ and a rotating speed of 150 rpm, and culturing for 3d to obtain a secondary strain;
1.2.3 three-stage liquid seed 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.
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;
1.3 extraction and refining of active ingredients in the final fermentation
1.3.1 Pretreatment of final fermentate
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.
Extraction of
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 2000rpm/min after extraction, concentrating supernatant at 100deg.C to 1/2 of original volume to obtain concentrated dealcoholized solution, recovering methanol, and refining by chromatography.
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 mu m, 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 20 to 70: gradient elution is carried out according to the proportion of 80-30, and a target peak with the molecular weight of 583 on a mass spectrum detector is collected; concentrating the collected matter below 100deg.C to viscous state, and drying at 170deg.C; the cyclophosphamide-pro-valyl-isoleucyl-leucinide was obtained as a white powder.
The structural identification result shows that the structural formula of the ring-color-propyl-valyl-isoleucyl-leucinyl peptide is as follows:
The activity measurement result shows that the semi-inhibition concentration (IC 50) of the cyclophilin-propyl-valyl-isoleucyl-leucinide on liver cancer cells HepG2 is as follows: 54.19 The half inhibitory concentration (IC 50) of α -glucosidase in μg/mL is: 63.64 μg/mL.
Example 2
The preparation method of the cyclophosphamide-propyl-valyl-isoleucyl-leucinide with hepatoma cytotoxicity and alpha-glucosidase inhibitory activity comprises the following steps:
2.1 selection of frog feces mould seed
The frog manure mould is rana spinosa manure mould;
2.2 Strain culture
The culture method is liquid culture, and comprises the following specific procedures:
2.2.1 slant seed culture
Inoculating the preserved frog-faecal fungus strain into potato agar (PDA) slant culture medium, and culturing at 36 deg.C for 8d to obtain first-class strain;
2.2.2 two-stage liquid seed culture
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 30.0 g/L, peptone 6.0 g/L, yeast extract 4.0g/L, potassium chloride (KCl) 0.3g/L, magnesium sulfate heptahydrate (MgSO 4.7H2 O) 0.3g/L, and potassium dihydrogen phosphate (KH 2PO4) 2.0 g/L.
Adding 50% of the volume of the triangular flask into the triangular flask of 1000 ml, inoculating 3 inclined plane strains into each triangular flask, placing the inoculated strains into a constant-temperature shaking incubator at 36 ℃ and the rotating speed of 250 rpm, and culturing for 7d to obtain the secondary strain.
Three-stage liquid seed 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.
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.
Extraction and refinement of active principles in the final fermentation
2.3.1 Pretreatment of final fermentate
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.
Extraction of
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 12000rpm/min after extraction, concentrating supernatant at 40deg.C to 1/10 of original volume to obtain concentrated dealcoholized solution, recovering ethanol, and refining by chromatography.
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 mu m, 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 20 to 70: gradient elution is carried out according to the proportion of 80-30, and a target peak with the molecular weight of 583 on a mass spectrum detector is collected; concentrating the collected matter below 30deg.C to viscous state, and drying at-30deg.C; the cyclic-propyl-valyl-isoleucyl-leucinide was obtained as a white powder.
The structural identification result shows that the structural formula of the cyclophospha-propyl-valyl-isoleucyl-leucinyl peptide is as follows:
the activity measurement result shows that the semi-inhibition concentration (IC 50) of the cyclophilin-propyl-valyl-isoleucyl-leucinide on liver cancer cells HepG2 is as follows: 54.19 The half inhibitory concentration (IC 50) of α -glucosidase in μg/mL is: 63.64 μg/mL.
Example 3:
the preparation method of the cyclophosphamide-propyl-valyl-isoleucyl-leucinide with hepatoma cytotoxicity and alpha-glucosidase inhibitory activity comprises the following steps:
3.1 selection of frog feces mould seed
The selected strain is the solid spore frog manure mould;
3.2 Strain culture
The culture method is liquid-solid mixed culture, and comprises the following specific procedures:
3.2.1 slant seed culture
Inoculating the preserved frog-faecal fungus strain into potato agar (PDA) slant culture medium, and culturing at 30deg.C for 6d to obtain first-class strain;
3.2.2 two-stage liquid seed culture
Inoculating the first-level strain to a liquid shake flask culture medium for culture;
Liquid shake flask medium: 35.0 g/L glucose, 20.0 g/L malt extract, 4.0 g/L peptone, 2.0g/L yeast extract powder, 0.3g/L potassium chloride (KCl), 0.3g/L magnesium sulfate heptahydrate (MgSO 4.7H2 O), 1.0 g/L potassium dihydrogen phosphate (KH 2PO4).
Adding 40% of liquid culture medium into 500 ml triangular flasks, inoculating 2 inclined plane strains into each triangular flask, placing into a constant temperature shaking incubator at 30deg.C and rotation speed of 200 rpm, and culturing for 5d to obtain secondary strain.
Three-stage liquid seed 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.
Four-stage culture
The four-stage culture is solid culture: mixing the three-stage liquid strain into a solid culture medium, wherein the inoculation amount is 3% of the solid material amount, 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: 25.0 g/L glucose, 10.0g/L malt extract, 2.0 g/L peptone, 1.0g/L yeast extract, 0.3g/L potassium chloride (KCl), 0.3g/L magnesium sulfate heptahydrate (MgSO 4.7H2 O), 0.3g/L potassium dihydrogen phosphate (KH 2PO4); agar 15.0 g/L.
Extraction and refinement of active principles in the final fermentation
3.3.1 Pretreatment of final fermentate
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.
Extraction of
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;
3.3.3 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 mu m, 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 20 to 70: gradient elution is carried out according to the proportion of 80-30, and a target peak with the molecular weight of 583 on a mass spectrum detector is collected; concentrating the collected matter at below 40deg.C to viscous state, and drying at 10deg.C; the cyclic-propyl-valyl-isoleucyl-leucinide was obtained as a white powder.
The structural identification result shows that the structural formula of the cyclophospha-propyl-valyl-isoleucyl-leucinyl peptide is as follows:
the activity measurement result shows that the semi-inhibition concentration (IC 50) of the cyclophilin-propyl-valyl-isoleucyl-leucinide on liver cancer cells HepG2 is as follows: 54.19 The half inhibitory concentration (IC 50) of α -glucosidase in μg/mL is: 63.64 μg/mL.
Example 4:
the preparation method of the cyclophosphamide-propyl-valyl-isoleucyl-leucinide with hepatoma cytotoxicity and alpha-glucosidase inhibitory activity comprises the following steps:
4.1 selection of frog feces mould seed
The used frog-feces mold is frog-raw frog-feces mold;
4.2 Strain culture
The culture method is liquid-solid mixed culture, and comprises the following specific procedures:
4.2.1 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;
4.2.2 two-stage liquid seed culture
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 25.0 g/L, peptone 3.0g/L, yeast extract 3.0g/L, potassium chloride (KCl) 0.3g/L, magnesium sulfate heptahydrate (MgSO 4.7H2 O) 0.3g/L, and potassium dihydrogen phosphate (KH 2PO4) 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 constant temperature shaking incubator at 30deg.C and rotation speed of 180 rpm, and culturing for 4d to obtain 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 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.
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 30.0 g/L, peptone 6.0 g/L, yeast extract 4.0g/L, potassium chloride (KCl) 0.3g/L, magnesium sulfate heptahydrate (MgSO 4.7H2 O) 0.3g/L, potassium dihydrogen phosphate (KH 2PO4) 2.0 g/L, and agar 30.0 g/L.
Extraction and refinement of active principles in the final fermentation
4.3.1 Pretreatment of final fermentate
The final fermentation product of the solid is dried to water content of 10% at 130 ℃, and then crushed to 120 meshes for standby.
Extraction of
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;
4.3.3 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 mu m, 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 20 to 70: gradient elution is carried out according to the proportion of 80-30, and a target peak with the molecular weight of 583 on a mass spectrum detector is collected; concentrating the collected matter below 100deg.C to viscous state, and drying at 170deg.C; the cyclic-propyl-valyl-isoleucyl-leucinide was obtained as a white powder.
The structural identification result shows that the structural formula of the cyclophospha-propyl-valyl-isoleucyl-leucinyl peptide is as follows:
the activity measurement result shows that the semi-inhibition concentration (IC 50) of the cyclophilin-propyl-valyl-isoleucyl-leucinide on liver cancer cells HepG2 is as follows: 54.19 The half inhibitory concentration (IC 50) of α -glucosidase in μg/mL is: 63.64 μg/mL.
Claims (1)
1. A cyclic color-pro-valyl-isoleucyl-leucinyl peptide having hepatoma cytotoxicity and α -glucosidase inhibitory activity, characterized in that: the structural formula of the cyclic-propyl-valyl-isoleucyl-leucinyl peptide is as follows:
；
the cyclic-propyl-valyl-isoleucyl-leupeptin is fifteen-membered cyclic pentapeptide formed by connecting tryptophan, alanine, valine, isoleucyl and leucine through an amide bond;
The cyclophosphamide-propyl-valyl-isoleucyl-leucinide has liver cancer cytotoxicity and alpha-glucosidase inhibitory activity; the semi-inhibitory concentration IC 50 for hepatoma cell HepG2 was: 54.19 Mu g/mL, semi-inhibitory concentration IC 50 of alpha-glucosidase: 63.64 μg/mL.
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