CN115651069B - Cyclic color-threo-tyr-valyl-phenylpropanoid with hepatoma cytotoxicity and alpha-glucosidase inhibitory activity and preparation method thereof - Google Patents
Cyclic color-threo-tyr-valyl-phenylpropanoid with hepatoma cytotoxicity and alpha-glucosidase inhibitory activity and preparation method thereof Download PDFInfo
- Publication number
- CN115651069B CN115651069B CN202211482981.9A CN202211482981A CN115651069B CN 115651069 B CN115651069 B CN 115651069B CN 202211482981 A CN202211482981 A CN 202211482981A CN 115651069 B CN115651069 B CN 115651069B
- Authority
- CN
- China
- Prior art keywords
- threo
- valyl
- phenylpropanoid
- tyr
- alpha
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 102100024295 Maltase-glucoamylase Human genes 0.000 title claims abstract description 37
- 108010028144 alpha-Glucosidases Proteins 0.000 title claims abstract description 37
- 230000002401 inhibitory effect Effects 0.000 title claims abstract description 26
- 231100000135 cytotoxicity Toxicity 0.000 title claims abstract description 24
- 230000003013 cytotoxicity Effects 0.000 title claims abstract description 24
- 206010073071 hepatocellular carcinoma Diseases 0.000 title claims abstract description 20
- 125000004122 cyclic group Chemical group 0.000 title claims abstract description 6
- 238000002360 preparation method Methods 0.000 title abstract description 12
- 201000007270 liver cancer Diseases 0.000 claims abstract description 12
- 208000014018 liver neoplasm Diseases 0.000 claims abstract description 12
- COLNVLDHVKWLRT-QMMMGPOBSA-N L-phenylalanine Chemical compound OC(=O)[C@@H](N)CC1=CC=CC=C1 COLNVLDHVKWLRT-QMMMGPOBSA-N 0.000 claims abstract description 4
- AYFVYJQAPQTCCC-GBXIJSLDSA-N L-threonine Chemical compound C[C@@H](O)[C@H](N)C(O)=O AYFVYJQAPQTCCC-GBXIJSLDSA-N 0.000 claims abstract description 4
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 claims abstract description 4
- QIVBCDIJIAJPQS-VIFPVBQESA-N L-tryptophane Chemical compound C1=CC=C2C(C[C@H](N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-VIFPVBQESA-N 0.000 claims abstract description 3
- KZSNJWFQEVHDMF-BYPYZUCNSA-N L-valine Chemical compound CC(C)[C@H](N)C(O)=O KZSNJWFQEVHDMF-BYPYZUCNSA-N 0.000 claims abstract description 3
- AYFVYJQAPQTCCC-UHFFFAOYSA-N Threonine Natural products CC(O)C(N)C(O)=O AYFVYJQAPQTCCC-UHFFFAOYSA-N 0.000 claims abstract description 3
- 239000004473 Threonine Substances 0.000 claims abstract description 3
- QIVBCDIJIAJPQS-UHFFFAOYSA-N Tryptophan Natural products C1=CC=C2C(CC(N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-UHFFFAOYSA-N 0.000 claims abstract description 3
- KZSNJWFQEVHDMF-UHFFFAOYSA-N Valine Natural products CC(C)C(N)C(O)=O KZSNJWFQEVHDMF-UHFFFAOYSA-N 0.000 claims abstract description 3
- COLNVLDHVKWLRT-UHFFFAOYSA-N phenylalanine Natural products OC(=O)C(N)CC1=CC=CC=C1 COLNVLDHVKWLRT-UHFFFAOYSA-N 0.000 claims abstract description 3
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 claims abstract description 3
- 239000004474 valine Substances 0.000 claims abstract description 3
- 229950003188 isovaleryl diethylamide Drugs 0.000 claims description 4
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 4
- 238000000605 extraction Methods 0.000 abstract description 22
- 230000000694 effects Effects 0.000 abstract description 18
- 238000012258 culturing Methods 0.000 abstract description 15
- 239000000843 powder Substances 0.000 abstract description 12
- 150000001875 compounds Chemical class 0.000 abstract description 7
- 239000003814 drug Substances 0.000 abstract description 7
- 230000005764 inhibitory process Effects 0.000 abstract description 7
- 108010069514 Cyclic Peptides Proteins 0.000 abstract description 2
- 102000001189 Cyclic Peptides Human genes 0.000 abstract description 2
- 239000007788 liquid Substances 0.000 description 54
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 42
- 238000000855 fermentation Methods 0.000 description 37
- 230000004151 fermentation Effects 0.000 description 37
- 239000001963 growth medium Substances 0.000 description 33
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 24
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 description 24
- 239000000047 product Substances 0.000 description 22
- 239000007787 solid Substances 0.000 description 21
- 239000000945 filler Substances 0.000 description 15
- 238000011218 seed culture Methods 0.000 description 15
- 238000005273 aeration Methods 0.000 description 14
- 238000000746 purification Methods 0.000 description 14
- 238000000034 method Methods 0.000 description 13
- 238000004366 reverse phase liquid chromatography Methods 0.000 description 12
- 238000012807 shake-flask culturing Methods 0.000 description 12
- 210000004027 cell Anatomy 0.000 description 10
- 238000011081 inoculation Methods 0.000 description 10
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 9
- 239000008103 glucose Substances 0.000 description 9
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 9
- 229920001817 Agar Polymers 0.000 description 8
- 229910019142 PO4 Inorganic materials 0.000 description 8
- 239000001888 Peptone Substances 0.000 description 8
- 108010080698 Peptones Proteins 0.000 description 8
- 239000008272 agar Substances 0.000 description 8
- 229940041514 candida albicans extract Drugs 0.000 description 8
- 239000000284 extract Substances 0.000 description 8
- 210000003608 fece Anatomy 0.000 description 8
- 238000009630 liquid culture Methods 0.000 description 8
- 238000011068 loading method Methods 0.000 description 8
- WRUGWIBCXHJTDG-UHFFFAOYSA-L magnesium sulfate heptahydrate Chemical compound O.O.O.O.O.O.O.[Mg+2].[O-]S([O-])(=O)=O WRUGWIBCXHJTDG-UHFFFAOYSA-L 0.000 description 8
- 229940061634 magnesium sulfate heptahydrate Drugs 0.000 description 8
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 8
- 235000019796 monopotassium phosphate Nutrition 0.000 description 8
- 235000019319 peptone Nutrition 0.000 description 8
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 description 8
- 239000001103 potassium chloride Substances 0.000 description 8
- 235000011164 potassium chloride Nutrition 0.000 description 8
- 238000007670 refining Methods 0.000 description 8
- 239000012138 yeast extract Substances 0.000 description 8
- 239000004480 active ingredient Substances 0.000 description 7
- 239000012528 membrane Substances 0.000 description 7
- 238000000926 separation method Methods 0.000 description 7
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 6
- 241000233866 Fungi Species 0.000 description 6
- 238000001035 drying Methods 0.000 description 6
- 238000001914 filtration Methods 0.000 description 6
- 239000002609 medium Substances 0.000 description 6
- 241000235579 Basidiobolus Species 0.000 description 5
- 230000005526 G1 to G0 transition Effects 0.000 description 5
- 244000061456 Solanum tuberosum Species 0.000 description 5
- 235000002595 Solanum tuberosum Nutrition 0.000 description 5
- 229940079593 drug Drugs 0.000 description 5
- 238000010828 elution Methods 0.000 description 5
- 238000004519 manufacturing process Methods 0.000 description 5
- 238000001819 mass spectrum Methods 0.000 description 5
- 239000000401 methanolic extract Substances 0.000 description 5
- 239000002245 particle Substances 0.000 description 5
- 239000000126 substance Substances 0.000 description 5
- 206010028980 Neoplasm Diseases 0.000 description 4
- 241000270959 Pelophylax nigromaculatus Species 0.000 description 4
- 201000011510 cancer Diseases 0.000 description 4
- 238000004587 chromatography analysis Methods 0.000 description 4
- 238000012136 culture method Methods 0.000 description 4
- 238000005259 measurement Methods 0.000 description 4
- 230000008569 process Effects 0.000 description 4
- 208000001072 type 2 diabetes mellitus Diseases 0.000 description 4
- 238000009423 ventilation Methods 0.000 description 4
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- 150000001720 carbohydrates Chemical class 0.000 description 3
- 229910052799 carbon Inorganic materials 0.000 description 3
- 239000000706 filtrate Substances 0.000 description 3
- 238000004108 freeze drying Methods 0.000 description 3
- 239000010871 livestock manure Substances 0.000 description 3
- 238000002156 mixing Methods 0.000 description 3
- 238000010298 pulverizing process Methods 0.000 description 3
- 239000011343 solid material Substances 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- 108010087230 Sincalide Proteins 0.000 description 2
- 238000002835 absorbance Methods 0.000 description 2
- 239000002246 antineoplastic agent Substances 0.000 description 2
- 229940041181 antineoplastic drug Drugs 0.000 description 2
- 235000021257 carbohydrate digestion Nutrition 0.000 description 2
- 238000010609 cell counting kit-8 assay Methods 0.000 description 2
- YJGVMLPVUAXIQN-UHFFFAOYSA-N epipodophyllotoxin Natural products COC1=C(OC)C(OC)=CC(C2C3=CC=4OCOC=4C=C3C(O)C3C2C(OC3)=O)=C1 YJGVMLPVUAXIQN-UHFFFAOYSA-N 0.000 description 2
- 239000002054 inoculum Substances 0.000 description 2
- 229920001542 oligosaccharide Polymers 0.000 description 2
- 150000002482 oligosaccharides Chemical class 0.000 description 2
- YJGVMLPVUAXIQN-XVVDYKMHSA-N podophyllotoxin Chemical compound COC1=C(OC)C(OC)=CC([C@@H]2C3=CC=4OCOC=4C=C3[C@H](O)[C@@H]3[C@@H]2C(OC3)=O)=C1 YJGVMLPVUAXIQN-XVVDYKMHSA-N 0.000 description 2
- 229960001237 podophyllotoxin Drugs 0.000 description 2
- YVCVYCSAAZQOJI-UHFFFAOYSA-N podophyllotoxin Natural products COC1=C(O)C(OC)=CC(C2C3=CC=4OCOC=4C=C3C(O)C3C2C(OC3)=O)=C1 YVCVYCSAAZQOJI-UHFFFAOYSA-N 0.000 description 2
- 239000013641 positive control Substances 0.000 description 2
- 230000005180 public health Effects 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- IZTQOLKUZKXIRV-YRVFCXMDSA-N sincalide Chemical compound C([C@@H](C(=O)N[C@@H](CCSC)C(=O)NCC(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC=1C=CC=CC=1)C(N)=O)NC(=O)[C@@H](N)CC(O)=O)C1=CC=C(OS(O)(=O)=O)C=C1 IZTQOLKUZKXIRV-YRVFCXMDSA-N 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 229940077274 Alpha glucosidase inhibitor Drugs 0.000 description 1
- 210000002237 B-cell of pancreatic islet Anatomy 0.000 description 1
- 241001340097 Copromyces Species 0.000 description 1
- 206010059866 Drug resistance Diseases 0.000 description 1
- 241000238631 Hexapoda Species 0.000 description 1
- 238000005481 NMR spectroscopy Methods 0.000 description 1
- 241001560470 Quasipaa spinosa Species 0.000 description 1
- 241000269435 Rana <genus> Species 0.000 description 1
- 101150006914 TRP1 gene Proteins 0.000 description 1
- LVTKHGUGBGNBPL-UHFFFAOYSA-N Trp-P-1 Chemical compound N1C2=CC=CC=C2C2=C1C(C)=C(N)N=C2C LVTKHGUGBGNBPL-UHFFFAOYSA-N 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 239000013543 active substance Substances 0.000 description 1
- 239000003888 alpha glucosidase inhibitor Substances 0.000 description 1
- 238000012443 analytical study Methods 0.000 description 1
- 230000006907 apoptotic process Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000000975 bioactive effect Effects 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 230000023852 carbohydrate metabolic process Effects 0.000 description 1
- 230000004663 cell proliferation Effects 0.000 description 1
- 238000002784 cytotoxicity assay Methods 0.000 description 1
- 231100000263 cytotoxicity test Toxicity 0.000 description 1
- 238000001212 derivatisation Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000011141 high resolution liquid chromatography Methods 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 239000003112 inhibitor Substances 0.000 description 1
- 230000003914 insulin secretion Effects 0.000 description 1
- 230000000968 intestinal effect Effects 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- 150000002611 lead compounds Chemical class 0.000 description 1
- 230000037356 lipid metabolism Effects 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 238000004895 liquid chromatography mass spectrometry Methods 0.000 description 1
- 238000004949 mass spectrometry Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000002207 metabolite Substances 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 210000000110 microvilli Anatomy 0.000 description 1
- 150000002772 monosaccharides Chemical class 0.000 description 1
- 244000052769 pathogen Species 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 230000022558 protein metabolic process Effects 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 238000012827 research and development Methods 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 239000002689 soil Substances 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 150000008494 α-glucosides Chemical class 0.000 description 1
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention relates to a cyclochrome-threo-Tyr-valyl-phenylpropanoid (Trp-Thr-Tyr-Val-Phe cyclic peptide) with hepatoma cytotoxicity and alpha-glucosidase inhibition activity and a preparation method thereof, belonging to the technical field of extraction and preparation of biological products. The cyclic-threo-tyr-valyl-phenylpropanoid is fifteen-membered cyclic pentapeptide formed by connecting tryptophan, threonine, tyrosine, valine and phenylalanine 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 cyclophosphamide-threo-tyrosin-valyl-phenylpropanoid is white powder, and the semi-inhibition concentration IC 50 of the cycloparaffin-threo-tyrosin-valyl-phenylpropanoid on liver cancer cells HepG2 is as follows: 62.30 μg/mL, half inhibitory concentration IC 50 of alpha-glucosidase: 60.42 μg/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-threo-Tyr-valyl-phenylpropanoid (Trp-Thr-Tyr-Val-Phe cyclopeptide) with hepatoma cytotoxicity and alpha-glucosidase inhibitory activity.
Background
The cyclophilin-threo-tyro-valyl-phenylpropanoid is a bioactive substance with hepatoma cytotoxicity and alpha-glucosidase inhibitory activity, which is isolated from a culture of frog-faecalis (Basidiobolus sp.).
The Rana Nigromaculata (Basidiobolus sp.) is fungus of Rana Nigromaculata genus of Rana Nigromaculata family of Rana of Pelaromycetales order Pelaromycetales; 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 cyclophosphamide-threo-tyr-valyl-phenylpropanoid which has 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-threo-tyro-valyl peptide extracted from the frog-like copromyces 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-threo-tyr-valyl-phenylpropanoid with hepatoma cytotoxicity and alpha-glucosidase inhibitory activity is as follows:
The cyclic-threo-tyr-valyl-phenylpropanoid is fifteen-membered cyclic pentapeptide formed by connecting tryptophan, threonine, tyrosine, valine and phenylalanine through an amide bond;
The cyclophosphamide-threo-tyr-valyl peptide has liver cancer cytotoxicity and alpha-glucosidase inhibitory activity;
The half inhibitory concentration (IC 50) on hepatoma cell HepG2 was: 62.30 μg/mL, half inhibitory concentration of α -glucosidase (IC 50) was: 60.42 μg/mL.
The preparation method of the cyclophosphamide-threo-tyr-valyl-phenylpropanoid with hepatoma cytotoxicity and alpha-glucosidase inhibitory activity comprises the following steps:
(1) Strain culture
Culturing Rana Nigromaculata (Basidiobolus sp.) by slant strain, culturing secondary liquid seed, culturing tertiary liquid seed and quaternary culturing to obtain final fermented product of liquid or final fermented product of solid;
The frog-excrement mould (Basidiobolus sp.) is one of frog-raw frog-excrement mould, solid spore frog-excrement mould or schizospore frog-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 powder of cyclophosphamide-threo-tyr-valyl-phenylpropanoid.
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 30-50% of the volume of the triangular flask into 100ml or more triangular flasks, inoculating 1-3 inclined plane strains into each triangular flask, and culturing for 3-7 d in a constant-temperature shaking incubator at 25-36 ℃ and with 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, 1.0-4.0 g/L yeast extract powder, 0.3-2.0 g/L potassium chloride (KCl), 0.3-2.0 g/L magnesium sulfate heptahydrate (MgSO 4.7H2 O) and 0.3-2.0 g/L potassium dihydrogen phosphate (KH 2PO4);
(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 697Da 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-tyr-valyl-phenylpropanoid is obtained as a white powder.
The analytical study of the present invention is described as follows:
1. Screening research shows that the frog-faecalis (Basidiobolus sp.) 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 450nm, and calculating to obtain the extract with the semi-inhibition concentration (IC 50) of HepG2 liver cancer cells: 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) Determination of cytotoxicity of cyclophilin-threo-tyr-valyl-phenylpropanoid on hepatoma cell 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 450nm, and calculating to obtain the extract with the semi-inhibition concentration (IC 50) of HepG2 liver cancer cells: 62.30 μg/mL.
(2) Determination of alpha-glucosidase inhibitory Activity of cyclophosphamide-threo-tyr-valyl peptide: 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: 60.42 μg/mL.
3. Chemical structure identification of active compounds
High resolution liquid chromatography mass spectrometry analysis showed purified active compound positive ion 697.3346 (m+h +)、719.3165(M+Na+), calculated molecular formula C 38H44N6O7.
The nmr analysis data are shown in the following table:
| unit | carbon | δC,type | δH,(J in Hz) | unit | carbon | δC,type | δH,(J in Hz) |
| Trp | 1 | 172.0,C | 18a | 36.0,CH2 | 2.70,dd | ||
| 2 | 56.6,CH | 4.10,m | 18b | 2.79,dd | |||
| 3 | 27.2,CH2 | 2.66,dd(15.3,5.4) | 19 | 128.2,C | |||
| 2.84,dd(15.3,8.8) | 20 | 130.3,CH | 6.97,d(8.4) | ||||
| 4 | 110.5,C | 21 | 115.5,CH | 6.60,d(8.4) | |||
| 5 | 123.7,CH | 6.91,s | 22 | 156.4,C | |||
| NH | 10.73,s | 17-NH | 8.45,d(7.6) | ||||
| 6 | 136.6,C | Val | 23 | 170.7,C | |||
| 7 | 111.8,CH | 7.27,d(7.8) | 24 | 58.8,CH | 3.86,t(7.6) | ||
| 8 | 121.4,CH | 7.00,t(7.4) | 25 | 31.3,CH | 1.78,m | ||
| 9 | 118.8,CH | 6.93,t(7.4) | 26 | 19.0,CH3 | 0.57,d(6.9) | ||
| 10 | 118.5,CH | 7.38,d(7.8) | 27 | 19.1,CH3 | 0.62,d(6.9) | ||
| 11 | 127.6,C | 24-NH | 8.14,d(7.5) | ||||
| 2-NH | 8.58,br s | Phe | 28 | 171.4,C | |||
| Thr | 12 | 171.4,C | 29 | 58.1,CH | 4.19,dt(8.3,8.5) | ||
| 13 | 58.7,CH | 4.07,dd(7.5) | 30 | 37.5,CH2 | 2.95,d(8.3) | ||
| 14 | 68.4,CH | 3.71,m | 31 | 138.2,C | |||
| 15 | 20.8,CH3 | 0.93,d(6.3) | 32 | 129.7,CH | 7.12,d(7.3) | ||
| 13-NH | 7.26,d(7.6) | 33 | 128.6,CH | 7.19,dd(7.3,7.3) | |||
| 14-OH | 4.83,s | 34 | 126.8,CH | 7.14,t(7.2) | |||
| Tyr | 16 | 172.2,C | 29-NH | 8.38,d(8.5) | |||
| 17 | 57.8,CH | 4.11,m |
The active compound structure obtained by separation and purification is cycloparaffin-threo-tyro-valyl-phenylpropanoid, 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-threo-tyro-valyl-phenylpropanoid 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 cyclophilin-threo-tyr-valyl-phenylpropanoid is a novel framework, can be used as a drug lead compound for derivatization and transformation, and can be used for creating compounds with more activities and stronger activity.
3. The cyclophosphamide-threo-tyr-valyl-phenylpropanoid of the present invention may 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-threo-tyr-valyl-phenylpropanoid 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, 2.0g/L peptone, 1.0g/L yeast extract powder, 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 100ml of triangular flask, inoculating 1 inclined plane strain to each triangular flask, placing the triangular flask into a constant-temperature shaking incubator at 25 ℃ and rotating at 150rpm, 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.
1.2.4 Four-stage culture
The four-stage culture adopts liquid culture: inoculating the three-level strain to a 50L fermentation tank, wherein the liquid loading amount is 50% of the tank volume, the inoculation amount is 3%, the ventilation is 1:1 (v/v) according to the volume ratio, the pressure is 0.1MP, and the constant-temperature ventilation culture is carried out for 5 days at 25 ℃ to obtain the final fermentation product of the liquid; the culture medium in the fermentation tank is the same as the liquid shake flask culture medium;
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.
1.3.2 Extraction
Extracting the dried mycelia with methanol; the feed-liquid ratio is 1:2 (W: V), the ultrasonic power is 10KHz, and the extraction time is 10 minutes; centrifuging at 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.
1.3.3 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 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 697Da on a mass spectrum detector is collected; concentrating the collected matter below 100deg.C to viscous state, and drying at 170deg.C; the cycloparaffin-threo-tyr-valyl-phenylpropanoid is obtained as a white powder.
The structural identification result shows that the structural formula of the cyclophosphamide-threo-tyr-valyl-phenylpropanoid is as follows:
The activity measurement result shows that the semi-inhibition concentration (IC 50) of the cyclophilin-threo-tyr-valyl-phenylpropanoid on liver cancer cells HepG2 is as follows: 62.30 μg/mL, half inhibitory concentration of α -glucosidase (IC 50) was: 60.42 μg/mL.
Example 2
The preparation method of the cyclophosphamide-threo-tyr-valyl-phenylpropanoid 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 50.0g/L, malt extract 30.0g/L, peptone 6.0g/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) 2.0g/L.
Adding 50% of the volume of the triangular flask into 1000ml of the triangular flask, inoculating 3 inclined plane strains into each triangular flask, placing the triangular flask into a constant-temperature shaking incubator at 36 ℃ and rotating at 250rpm after inoculation, and culturing for 7d to obtain a secondary strain.
2.2.3 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.
2.2.4 Four-stage cultivation
The four-stage culture is liquid culture: inoculating the three-level strain to a fermentation tank with the volume of more than or equal to 500L, wherein the liquid loading amount is 80% of the tank volume, the inoculum size is 10%, the aeration amount is 1:2 (v/v) in volume ratio, the pressure is 0.2MP, and the constant-temperature aeration culture is carried out for 15 days at 36 ℃ to obtain the final fermentation product of the liquid; the culture medium in the fermentation tank is the same as the liquid shake flask culture medium.
2.3 Extraction and refining of active ingredients 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.
2.3.2 Extraction
Extracting the dried mycelium with ethanol at a feed-liquid ratio of 1:20 (W: V) and ultrasonic power of 100KHz for 200 min; centrifuging at 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.
2.3.3 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 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 697Da on a mass spectrum detector is collected; concentrating the collected matter below 30deg.C to viscous state, and drying at-30deg.C; the cyclic-threo-tyr-valyl-phenylpropanoid is obtained as a white powder.
The structural identification result shows that the structural formula of the cyclophosphamide-threo-tyr-valyl-phenylpropanoid is as follows:
The activity measurement result shows that the semi-inhibition concentration (IC 50) of the cyclophilin-threo-tyr-valyl-phenylpropanoid on liver cancer cells HepG2 is as follows: 62.30 μg/mL, half inhibitory concentration of α -glucosidase (IC 50) was: 60.42 μg/mL.
Example 3:
the preparation method of the cyclophosphamide-threo-tyr-valyl-phenylpropanoid 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: 35.0g/L glucose, 20.0g/L malt extract, 4.0g/L peptone, 2.0g/L yeast extract powder, 0.3g/L potassium chloride (KCl), 0.3g/L magnesium sulfate heptahydrate (MgSO 4.7H2 O), 1.0g/L potassium dihydrogen phosphate (KH 2PO4).
Adding 40% of liquid culture medium into 500ml triangular flask, inoculating 2 slant strains into each triangular flask, placing into a constant temperature shaking incubator at 30deg.C and rotation speed of 200rpm, and culturing for 5d to obtain secondary strain.
3.2.3 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.
3.2.4 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: 25.0g/L glucose, 10.0g/L malt extract, 2.0g/L peptone, 1.0g/L yeast extract powder, 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 15.0g/L.
3.3 Extraction and purification of the active ingredients 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.
3.3.2 Extraction
Extracting the dried and crushed final fermentation product with ethanol; the feed-liquid ratio is 1:2 (W: V), the ultrasonic power is 10KHz, and the extraction time is 10 minutes; filtering with 0.20 μm filter membrane after extraction; concentrating the filtrate to 1/2 of the original volume at 30deg.C to obtain concentrated dealcoholized solution, recovering ethanol, and refining the concentrated dealcoholized solution by further chromatography;
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 697Da 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-threo-tyr-valyl-phenylpropanoid is obtained as a white powder.
The structural identification result shows that the structural formula of the cyclophosphamide-threo-tyr-valyl-phenylpropanoid is as follows:
The activity measurement result shows that the semi-inhibition concentration (IC 50) of the cyclophilin-threo-tyr-valyl-phenylpropanoid on liver cancer cells HepG2 is as follows: 62.30 μg/mL, half inhibitory concentration of α -glucosidase (IC 50) was: 60.42 μg/mL.
Example 4:
the preparation method of the cyclophosphamide-threo-tyr-valyl-phenylpropanoid 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 40.0g/L, malt extract 25.0g/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) 1.2g/L.
Adding 40% of liquid culture medium into 500ml triangular flask, inoculating 1 slant strain into each triangular flask, placing into a constant temperature shaking incubator at 30deg.C and rotation speed of 180rpm, and culturing for 4d to obtain secondary strain.
4.2.3 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.
4.2.4 Four-stage culture
The four-stage culture adopts solid culture: mixing the three-stage liquid strain into a solid culture medium, wherein the inoculation amount is 10% of the solid material amount, and carrying out aeration culture at a constant temperature of 36 ℃ for 8 days to obtain a solid final fermentation product;
The solid culture medium is as follows: glucose 50.0g/L, malt extract 30.0g/L, peptone 6.0g/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) 2.0g/L, and agar 30.0g/L.
4.3 Extraction and refining of active ingredients 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.
4.3.2 Extraction
Extracting the dried and crushed final fermentation product with methanol at a feed-liquid ratio of 1:20 (W: V) and ultrasonic power of 100KHz for 200 min; filtering with 1.0 μm filter membrane after extraction; concentrating the filtrate at 40deg.C to 1/10 of the original volume to obtain concentrated dealcoholized solution, recovering methanol, and refining the concentrated dealcoholized solution by further chromatography;
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 697Da on a mass spectrum detector is collected; concentrating the collected matter below 100deg.C to viscous state, and drying at 170deg.C; the cyclic-threo-tyr-valyl-phenylpropanoid is obtained as a white powder.
The structural identification result shows that the structural formula of the cyclophosphamide-threo-tyr-valyl-phenylpropanoid is as follows:
The activity measurement result shows that the semi-inhibition concentration (IC 50) of the cyclophilin-threo-tyr-valyl-phenylpropanoid on liver cancer cells HepG2 is as follows: 62.30 μg/mL, half inhibitory concentration of α -glucosidase (IC 50) was: 60.42 μg/mL.
Claims (1)
1. A cyclic color-threo-tyr-valyl-phenylpropanoid having hepatoma cytotoxicity and α -glucosidase inhibitory activity, characterized by: the structural formula of the cyclic-color-threo-casein-valyl-phenylpropanoid is as follows:
；
The cyclic-threo-tyr-valyl-phenylpropanoid is fifteen-membered cyclic pentapeptide formed by connecting tryptophan, threonine, tyrosine, valine and phenylalanine through an amide bond;
The cyclophosphamide-threo-tyr-valyl peptide has liver cancer cytotoxicity and alpha-glucosidase inhibitory activity; the semi-inhibitory concentration IC 50 for hepatoma cell HepG2 was: 62.30 Mu g/mL, semi-inhibitory concentration IC 50 of alpha-glucosidase: 60.42 μg/mL.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211482981.9A CN115651069B (en) | 2022-11-24 | 2022-11-24 | Cyclic color-threo-tyr-valyl-phenylpropanoid with hepatoma cytotoxicity and alpha-glucosidase inhibitory activity and preparation method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211482981.9A CN115651069B (en) | 2022-11-24 | 2022-11-24 | Cyclic color-threo-tyr-valyl-phenylpropanoid with hepatoma cytotoxicity and alpha-glucosidase inhibitory activity and preparation method thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN115651069A CN115651069A (en) | 2023-01-31 |
| CN115651069B true CN115651069B (en) | 2024-04-30 |
Family
ID=85019592
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202211482981.9A Active CN115651069B (en) | 2022-11-24 | 2022-11-24 | Cyclic color-threo-tyr-valyl-phenylpropanoid with hepatoma cytotoxicity and alpha-glucosidase inhibitory activity and preparation method thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN115651069B (en) |
Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| AU2013203028A1 (en) * | 2009-01-12 | 2013-05-02 | Novartis Ag | Cna_B domain antigens in vaccines against gram positive bacteria |
| EP3323422A1 (en) * | 2016-11-22 | 2018-05-23 | Université de Strasbourg | New d-configured cateslytin peptide |
| CN111018953A (en) * | 2020-01-19 | 2020-04-17 | 安徽农业大学 | Cyclocasein-isoleucin-leucin-chromo-threonine with antifungal and free radical scavenging activity and preparation method thereof |
| CN111018954A (en) * | 2020-01-19 | 2020-04-17 | 安徽农业大学 | Cyclo-serine-valine-leucine peptide with antifungal and free radical scavenging activities and preparation method thereof |
| CN113264987A (en) * | 2021-05-31 | 2021-08-17 | 安徽农业大学 | Cyclochromo-threo-valine-isoleucin with antifungal and free radical scavenging activity and preparation method thereof |
| CN113307848A (en) * | 2021-05-31 | 2021-08-27 | 安徽农业大学 | Cyclo-serine-valine-isoleucyl-leucyl peptide with antifungal and free radical scavenging activities and preparation method thereof |
| CN113583094A (en) * | 2021-09-09 | 2021-11-02 | 皖北卫生职业学院 | Cyclo-valine-silk-isoleucin-leucin with antifungal and free radical scavenging activities and preparation method thereof |
-
2022
- 2022-11-24 CN CN202211482981.9A patent/CN115651069B/en active Active
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| AU2013203028A1 (en) * | 2009-01-12 | 2013-05-02 | Novartis Ag | Cna_B domain antigens in vaccines against gram positive bacteria |
| EP3323422A1 (en) * | 2016-11-22 | 2018-05-23 | Université de Strasbourg | New d-configured cateslytin peptide |
| CN111018953A (en) * | 2020-01-19 | 2020-04-17 | 安徽农业大学 | Cyclocasein-isoleucin-leucin-chromo-threonine with antifungal and free radical scavenging activity and preparation method thereof |
| CN111018954A (en) * | 2020-01-19 | 2020-04-17 | 安徽农业大学 | Cyclo-serine-valine-leucine peptide with antifungal and free radical scavenging activities and preparation method thereof |
| CN113264987A (en) * | 2021-05-31 | 2021-08-17 | 安徽农业大学 | Cyclochromo-threo-valine-isoleucin with antifungal and free radical scavenging activity and preparation method thereof |
| CN113307848A (en) * | 2021-05-31 | 2021-08-27 | 安徽农业大学 | Cyclo-serine-valine-isoleucyl-leucyl peptide with antifungal and free radical scavenging activities and preparation method thereof |
| CN113583094A (en) * | 2021-09-09 | 2021-11-02 | 皖北卫生职业学院 | Cyclo-valine-silk-isoleucin-leucin with antifungal and free radical scavenging activities and preparation method thereof |
Non-Patent Citations (2)
| Title |
|---|
| Cyclic pentapeptides with anti-inflammatory, cytotoxic or α-glucosidase inhibitory activities from Basidiobolus meristosporus;Cheng Zhao等;Phytochemistry;20230305;第209卷;1-6 * |
| 小桐子活性成分在农药方面的研究进展;夏咸松;浦恩堂;严胜骄;林军;;现代农药;20090410(第02期);全文 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN115651069A (en) | 2023-01-31 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN113264987B (en) | Cyclic color-threo-valyl-isoleucyl-leucinyl peptide with antifungal and free radical scavenging activities and preparation method thereof | |
| CN113307848B (en) | Cyclic color-silk-valyl-isoleucyl-leucinyl peptide with antifungal and free radical scavenging activities and preparation method thereof | |
| CN111018954B (en) | Cyclo-serine-valine-leucine peptide with antifungal and free radical scavenging activities and preparation method thereof | |
| CN111018953B (en) | Cyclocasein-isoleucyl-leucyl-tryptophyl-threo-peptide with antifungal and free radical scavenging activities and preparation method thereof | |
| CN110386860B (en) | Efficient extraction method of cannabidiol | |
| CN107721990B (en) | Marine fungus-derived isoindolinone compounds, preparation method thereof and application thereof in preparation of anti-inflammatory drugs | |
| CN113583094A (en) | Cyclo-valine-silk-isoleucin-leucin with antifungal and free radical scavenging activities and preparation method thereof | |
| CN114478565B (en) | Monascin A with glycosidase activity inhibiting and immunoregulatory activity and preparation method thereof | |
| CN111235036B (en) | Eurotium cristatum and method for separating and purifying diketopiperazine dimer from eurotium cristatum | |
| CN115651069B (en) | Cyclic color-threo-tyr-valyl-phenylpropanoid with hepatoma cytotoxicity and alpha-glucosidase inhibitory activity and preparation method thereof | |
| CN115636871B (en) | Cyclic color-threo-tyrosol-isoleucyl-phenylpropanoid with hepatoma cytotoxicity and alpha-glucosidase inhibitory activity and preparation method thereof | |
| CN115716865B (en) | Cyclic color-threo-tyr-valyl-leupeptin with hepatoma cytotoxicity and alpha-glucosidase inhibition activity and preparation method thereof | |
| CN115651068B (en) | Cyclic color-propyl-casein-isopropyl-phenyl propyl peptide with hepatoma cytotoxicity and alpha-glucosidase inhibitory activity and preparation method thereof | |
| CN115636870B (en) | Cyclic color-threo-valyl-leucinyl peptide with hepatoma cytotoxicity and alpha-glucosidase inhibitory activity and preparation method thereof | |
| CN115651067B (en) | Cyclic color-propyl-valyl-isoleucyl-leucinide with hepatoma cytotoxicity and alpha-glucosidase inhibitory activity and preparation method thereof | |
| CN116023443B (en) | Cyclochrome-silk-valyl-leucinyl peptide with hepatoma cytotoxicity and alpha-glucosidase inhibitory activity and preparation method thereof | |
| CN115536734B (en) | Cyclic color-propyl-valyl-leucins with hepatoma cytotoxicity and alpha-glucosidase inhibitory activity and preparation method thereof | |
| CN115636869B (en) | Cyclic stachyose isoleucyl-propyl peptide with hepatoma cytotoxicity and alpha-glucosidase inhibition activity and preparation method thereof | |
| CN115572324B (en) | Cyclic color-valyl-leu-phenylpropanoid with hepatoma cytotoxicity and alpha-glucosidase inhibitory activity and preparation method thereof | |
| CN115716864B (en) | Cyclic color-valyl-leucinyl peptide with hepatoma cytotoxicity and alpha-glucosidase inhibitory activity and preparation method thereof | |
| CN104278070B (en) | Method for improving content of ergosterol in liquid fermentation products of phellinus igniarius | |
| CN114292280A (en) | Monascus B with glycosidase inhibiting activity and immunoregulation activity and preparation method thereof | |
| CN112843026B (en) | Application of diphenyl ether compound in preparation of acetylcholinesterase inhibitor or medicament for treating Alzheimer disease | |
| CN114478564A (en) | Monascus C with glycosidase inhibiting activity and immunoregulation activity and preparation method thereof | |
| CN109880747B (en) | Preparation method of hirsutella sinensis artificial culture |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant |