CN115286590B - De-iron high-iron pigment compound, derivative thereof, pharmaceutical composition and application thereof - Google Patents
De-iron high-iron pigment compound, derivative thereof, pharmaceutical composition and application thereof Download PDFInfo
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- CN115286590B CN115286590B CN202210989927.7A CN202210989927A CN115286590B CN 115286590 B CN115286590 B CN 115286590B CN 202210989927 A CN202210989927 A CN 202210989927A CN 115286590 B CN115286590 B CN 115286590B
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- 150000001875 compounds Chemical class 0.000 title claims abstract description 79
- 239000000049 pigment Substances 0.000 title claims abstract description 65
- XEEYBQQBJWHFJM-UHFFFAOYSA-N iron Substances [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 title claims abstract description 57
- 229910052742 iron Inorganic materials 0.000 title claims abstract description 49
- 239000008194 pharmaceutical composition Substances 0.000 title claims abstract description 11
- 238000002360 preparation method Methods 0.000 claims abstract description 18
- 241000221638 Morchella Species 0.000 claims abstract description 16
- 238000000034 method Methods 0.000 claims abstract description 4
- 239000002904 solvent Substances 0.000 claims description 53
- 239000003960 organic solvent Substances 0.000 claims description 39
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 36
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 33
- 238000000926 separation method Methods 0.000 claims description 22
- 239000000284 extract Substances 0.000 claims description 20
- 238000004440 column chromatography Methods 0.000 claims description 19
- 150000002825 nitriles Chemical class 0.000 claims description 19
- 206010028980 Neoplasm Diseases 0.000 claims description 17
- 239000003480 eluent Substances 0.000 claims description 17
- 238000000605 extraction Methods 0.000 claims description 14
- 239000000287 crude extract Substances 0.000 claims description 12
- 238000010828 elution Methods 0.000 claims description 12
- 239000012046 mixed solvent Substances 0.000 claims description 12
- 239000003795 chemical substances by application Substances 0.000 claims description 11
- 239000000463 material Substances 0.000 claims description 11
- 238000004128 high performance liquid chromatography Methods 0.000 claims description 10
- 238000004062 sedimentation Methods 0.000 claims description 10
- 239000004480 active ingredient Substances 0.000 claims description 6
- 238000002156 mixing Methods 0.000 claims description 6
- 150000003839 salts Chemical class 0.000 claims description 5
- 239000012453 solvate Substances 0.000 claims description 5
- 240000002769 Morchella esculenta Species 0.000 claims description 4
- 235000002779 Morchella esculenta Nutrition 0.000 claims description 4
- 238000013375 chromatographic separation Methods 0.000 claims description 4
- 238000001556 precipitation Methods 0.000 claims description 4
- 239000002246 antineoplastic agent Substances 0.000 claims description 3
- 229940041181 antineoplastic drug Drugs 0.000 claims description 3
- 201000010897 colon adenocarcinoma Diseases 0.000 claims description 3
- 208000029742 colonic neoplasm Diseases 0.000 claims description 3
- 150000002576 ketones Chemical class 0.000 claims description 3
- 201000007270 liver cancer Diseases 0.000 claims description 3
- 208000014018 liver neoplasm Diseases 0.000 claims description 3
- 239000000546 pharmaceutical excipient Substances 0.000 claims description 2
- 238000004519 manufacturing process Methods 0.000 claims 1
- 210000004027 cell Anatomy 0.000 abstract description 12
- 230000000694 effects Effects 0.000 abstract description 11
- -1 iron ions Chemical class 0.000 abstract description 10
- 239000002994 raw material Substances 0.000 abstract description 7
- 230000000259 anti-tumor effect Effects 0.000 abstract description 5
- 239000003814 drug Substances 0.000 abstract description 4
- 230000004565 tumor cell growth Effects 0.000 abstract description 3
- NEAQRZUHTPSBBM-UHFFFAOYSA-N 2-hydroxy-3,3-dimethyl-7-nitro-4h-isoquinolin-1-one Chemical compound C1=C([N+]([O-])=O)C=C2C(=O)N(O)C(C)(C)CC2=C1 NEAQRZUHTPSBBM-UHFFFAOYSA-N 0.000 abstract description 2
- 239000002253 acid Substances 0.000 abstract description 2
- 229940079593 drug Drugs 0.000 abstract description 2
- 238000009776 industrial production Methods 0.000 abstract description 2
- 230000005764 inhibitory process Effects 0.000 abstract description 2
- 239000000797 iron chelating agent Substances 0.000 abstract description 2
- 229940075525 iron chelating agent Drugs 0.000 abstract description 2
- 230000037353 metabolic pathway Effects 0.000 abstract description 2
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 27
- 235000019441 ethanol Nutrition 0.000 description 17
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 15
- 238000012360 testing method Methods 0.000 description 9
- 238000002137 ultrasound extraction Methods 0.000 description 9
- 239000000243 solution Substances 0.000 description 8
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 6
- 230000012010 growth Effects 0.000 description 6
- 229910052739 hydrogen Inorganic materials 0.000 description 6
- 239000001257 hydrogen Substances 0.000 description 6
- 238000003756 stirring Methods 0.000 description 6
- 125000004169 (C1-C6) alkyl group Chemical group 0.000 description 5
- GHASVSINZRGABV-UHFFFAOYSA-N Fluorouracil Chemical compound FC1=CNC(=O)NC1=O GHASVSINZRGABV-UHFFFAOYSA-N 0.000 description 5
- 229960002949 fluorouracil Drugs 0.000 description 5
- 230000002401 inhibitory effect Effects 0.000 description 5
- 238000002835 absorbance Methods 0.000 description 4
- 238000001704 evaporation Methods 0.000 description 4
- 239000013641 positive control Substances 0.000 description 4
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 3
- KWYUFKZDYYNOTN-UHFFFAOYSA-M Potassium hydroxide Chemical compound [OH-].[K+] KWYUFKZDYYNOTN-UHFFFAOYSA-M 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 238000012258 culturing Methods 0.000 description 3
- 238000001514 detection method Methods 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- 238000002953 preparative HPLC Methods 0.000 description 3
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 2
- 208000000461 Esophageal Neoplasms Diseases 0.000 description 2
- 206010030155 Oesophageal carcinoma Diseases 0.000 description 2
- 206010061902 Pancreatic neoplasm Diseases 0.000 description 2
- 208000005718 Stomach Neoplasms Diseases 0.000 description 2
- GLLRIXZGBQOFLM-UHFFFAOYSA-N Xanthorin Natural products C1=C(C)C=C2C(=O)C3=C(O)C(OC)=CC(O)=C3C(=O)C2=C1O GLLRIXZGBQOFLM-UHFFFAOYSA-N 0.000 description 2
- 238000010521 absorption reaction Methods 0.000 description 2
- 239000005456 alcohol based solvent Substances 0.000 description 2
- 150000001412 amines Chemical class 0.000 description 2
- 239000007864 aqueous solution Substances 0.000 description 2
- 238000001035 drying Methods 0.000 description 2
- 201000004101 esophageal cancer Diseases 0.000 description 2
- 230000008020 evaporation Effects 0.000 description 2
- 235000013305 food Nutrition 0.000 description 2
- 206010017758 gastric cancer Diseases 0.000 description 2
- 150000004676 glycans Chemical class 0.000 description 2
- 230000009036 growth inhibition Effects 0.000 description 2
- 230000014759 maintenance of location Effects 0.000 description 2
- 208000015486 malignant pancreatic neoplasm Diseases 0.000 description 2
- 239000013642 negative control Substances 0.000 description 2
- 201000002528 pancreatic cancer Diseases 0.000 description 2
- 208000008443 pancreatic carcinoma Diseases 0.000 description 2
- 229920001282 polysaccharide Polymers 0.000 description 2
- 239000005017 polysaccharide Substances 0.000 description 2
- 238000010298 pulverizing process Methods 0.000 description 2
- 239000013049 sediment Substances 0.000 description 2
- 201000011549 stomach cancer Diseases 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 238000001644 13C nuclear magnetic resonance spectroscopy Methods 0.000 description 1
- 238000005160 1H NMR spectroscopy Methods 0.000 description 1
- VZSRBBMJRBPUNF-UHFFFAOYSA-N 2-(2,3-dihydro-1H-inden-2-ylamino)-N-[3-oxo-3-(2,4,6,7-tetrahydrotriazolo[4,5-c]pyridin-5-yl)propyl]pyrimidine-5-carboxamide Chemical compound C1C(CC2=CC=CC=C12)NC1=NC=C(C=N1)C(=O)NCCC(N1CC2=C(CC1)NN=N2)=O VZSRBBMJRBPUNF-UHFFFAOYSA-N 0.000 description 1
- 125000004493 2-methylbut-1-yl group Chemical group CC(C*)CC 0.000 description 1
- 125000005916 2-methylpentyl group Chemical group 0.000 description 1
- 125000005917 3-methylpentyl group Chemical group 0.000 description 1
- 241000228212 Aspergillus Species 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- 244000199866 Lactobacillus casei Species 0.000 description 1
- 235000013958 Lactobacillus casei Nutrition 0.000 description 1
- 244000187342 Lactobacillus casei ATCC 334 Species 0.000 description 1
- 235000003418 Lactobacillus casei ATCC 334 Nutrition 0.000 description 1
- 238000005481 NMR spectroscopy Methods 0.000 description 1
- 241000228143 Penicillium Species 0.000 description 1
- 239000007983 Tris buffer Substances 0.000 description 1
- PBCJIPOGFJYBJE-UHFFFAOYSA-N acetonitrile;hydrate Chemical compound O.CC#N PBCJIPOGFJYBJE-UHFFFAOYSA-N 0.000 description 1
- 230000006978 adaptation Effects 0.000 description 1
- 229910021529 ammonia Inorganic materials 0.000 description 1
- 150000003863 ammonium salts Chemical class 0.000 description 1
- 230000004663 cell proliferation Effects 0.000 description 1
- 230000003833 cell viability Effects 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- DQLATGHUWYMOKM-UHFFFAOYSA-L cisplatin Chemical compound N[Pt](N)(Cl)Cl DQLATGHUWYMOKM-UHFFFAOYSA-L 0.000 description 1
- 229960004316 cisplatin Drugs 0.000 description 1
- 239000012141 concentrate Substances 0.000 description 1
- 239000008367 deionised water Substances 0.000 description 1
- 229910021641 deionized water Inorganic materials 0.000 description 1
- 239000003085 diluting agent Substances 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 125000001495 ethyl group Chemical group [H]C([H])([H])C([H])([H])* 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 230000009950 gastric cancer growth Effects 0.000 description 1
- 125000004491 isohexyl group Chemical group C(CCC(C)C)* 0.000 description 1
- 125000001972 isopentyl group Chemical group [H]C([H])([H])C([H])(C([H])([H])[H])C([H])([H])C([H])([H])* 0.000 description 1
- 125000001449 isopropyl group Chemical group [H]C([H])([H])C([H])(*)C([H])([H])[H] 0.000 description 1
- 239000005453 ketone based solvent Substances 0.000 description 1
- 229940017800 lactobacillus casei Drugs 0.000 description 1
- GBMDVOWEEQVZKZ-UHFFFAOYSA-N methanol;hydrate Chemical compound O.OC GBMDVOWEEQVZKZ-UHFFFAOYSA-N 0.000 description 1
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 239000000178 monomer Substances 0.000 description 1
- 125000004108 n-butyl group Chemical group [H]C([H])([H])C([H])([H])C([H])([H])C([H])([H])* 0.000 description 1
- 125000001280 n-hexyl group Chemical group C(CCCCC)* 0.000 description 1
- 125000000740 n-pentyl group Chemical group [H]C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])* 0.000 description 1
- 239000011148 porous material Substances 0.000 description 1
- XAEFZNCEHLXOMS-UHFFFAOYSA-M potassium benzoate Chemical compound [K+].[O-]C(=O)C1=CC=CC=C1 XAEFZNCEHLXOMS-UHFFFAOYSA-M 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 239000006041 probiotic Substances 0.000 description 1
- 230000000529 probiotic effect Effects 0.000 description 1
- 235000018291 probiotics Nutrition 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 125000001436 propyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 229940043267 rhodamine b Drugs 0.000 description 1
- 125000002914 sec-butyl group Chemical group [H]C([H])([H])C([H])([H])C([H])(*)C([H])([H])[H] 0.000 description 1
- 229930000044 secondary metabolite Natural products 0.000 description 1
- 159000000000 sodium salts Chemical class 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 125000000999 tert-butyl group Chemical group [H]C([H])([H])C(*)(C([H])([H])[H])C([H])([H])[H] 0.000 description 1
- YNJBWRMUSHSURL-UHFFFAOYSA-N trichloroacetic acid Chemical compound OC(=O)C(Cl)(Cl)Cl YNJBWRMUSHSURL-UHFFFAOYSA-N 0.000 description 1
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
- 238000004506 ultrasonic cleaning Methods 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D259/00—Heterocyclic compounds containing rings having more than four nitrogen atoms as the only ring hetero atoms
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
- C07F15/00—Compounds containing elements of Groups 8, 9, 10 or 18 of the Periodic Table
- C07F15/02—Iron compounds
- C07F15/03—Sideramines; The corresponding desferri compounds
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- General Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Pharmacology & Pharmacy (AREA)
- Life Sciences & Earth Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The invention provides a de-ferriferrous pigment compound, a derivative thereof, a pharmaceutical composition and application thereof, and relates to the technical field of biological medicines. The iron-removing high-iron pigment compound with the structure shown in the formula I belongs to an iron chelating agent compound, and the hydroxamic acid structure in the structure can be chelated with iron ions so as to influence the related metabolic pathway of the iron ions in cells, so that the iron-removing high-iron pigment compound has good tumor cell growth inhibition activity and potential anti-tumor application prospect. The method separates and purifies the iron-removing high-iron pigment compound with the structure shown in the formula I from the Morchella for the first time, has high safety and effectiveness, and greatly expands the pharmaceutical economic value of the Morchella; the preparation method is simple to operate, low in cost and low in cost, and raw materials are cheap and easy to obtain, so that the preparation method is suitable for industrial production; in addition, the preparation method provided by the invention can avoid using a large amount of organic raw materials, and is environment-friendly.
Description
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to a de-iron high-iron pigment compound, a derivative thereof, a pharmaceutical composition and application thereof.
Background
The discovery of new structures and new sources of antitumor active compounds has been a hotspot in the field of pharmaceutical research. The iron-removing high iron pigments were first found in secondary metabolites of fungi of the genus Aspergillus, penicillium, etc. In recent years, researchers obtain the iron-removing high-iron pigment from the probiotic lactobacillus casei (Lactobacillus caseiATCC 334) which can inhibit the growth of gastric cancer cells, has better effect than that of a control medicament cisplatin and 5-fluorouracil, and has less influence on the growth of normal cells IEC-18 than that of the control medicament; the experimental result of the tumor allograft model further proves that the tumor allograft model has good anti-tumor effect. However, the iron-removing high-iron pigment compounds with anti-tumor effect still remain to be enriched.
Disclosure of Invention
In view of the above, the present invention aims to provide the desferriferrous iron pigment compound, the derivative, the pharmaceutical composition and the application thereof.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a de-ferriferrous pigment compound which has a structure shown in a formula I:
in the formula I, R 1 、R 2 And R is 3 Independently selected from-OH, -CH 2 OH, hydrogen or C1-C6 alkyl, and R 1 、R 1 And R is 3 Are not hydrogen at the same time;
R 4 、R 5 and R is 6 Independently selected from-OH, -CH 2 OH, hydrogen or C1-C6 alkyl.
Preferably, the de-iron high iron pigment compound has the following structure:
the invention provides a preparation method of the de-iron high-iron pigment compound, which comprises the following steps:
(1) Mixing Morchella esculenta with the extractant, and extracting to obtain extract; the extracting agent is water or an organic solvent-water mixed solvent, and the organic solvent in the extracting solvent comprises one or more of an alcohol solvent, a nitrile solvent and a ketone solvent;
(2) Carrying out sugar precipitation on the extract to obtain a sugar-removed crude extract;
(3) Separating the crude extract from the desugared extract by column chromatography to obtain a component Fr.2; the eluent adopted by the column chromatography separation is an organic solvent-water solvent, wherein the organic solvent comprises an alcohol solvent and/or a nitrile solvent, and the volume fraction of the organic solvent in the eluent is 0-100%;
(4) Subjecting the component Fr.2 to gel column chromatography separation to obtain a component Fr.2.2; the eluent for gel column chromatographic separation is an organic solvent-water mixed solvent, wherein the organic solvent comprises an alcohol solvent and/or a nitrile solvent, and the volume fraction of the organic solvent in the eluent is 30-60%;
(5) Separating the component Fr.2.2 by high performance liquid chromatography to obtain the de-iron high-iron pigment compound; the mobile phase adopted by the high performance liquid chromatography separation is an organic solvent-water mixed solvent, the organic solvent in the mobile phase comprises an alcohol solvent and/or a nitrile solvent, and the volume fraction of the organic solvent in the mobile phase is 4-20%.
Preferably, in the step (1), the ratio of the dry weight of Morchella to the volume of the extractant is 1kg: 2-20L;
the times of extraction are 2-5 times, and the time of single extraction is 1-6 hours.
Preferably, in the step (2), the solvent sedimentation agent for sugar sedimentation comprises water and an organic solvent, and the volume ratio of the water to the organic solvent is 1: (1.6-10), wherein the organic solvent comprises an alcohol solvent and/or a nitrile solvent;
the ratio of the dry weight of Morchella to the volume of the sedimentation agent is 1kg: 0.5-2.25L.
Preferably, in the step (3), the elution mode of the column chromatography separation is gradient elution, and the gradient elution is gradient elution by using organic solvent-water solvent with the volume fraction of the organic solvent of 0%, 30-59%, 60-90% and 100% in sequence.
The invention provides a derivative of a desferriferrous pigment compound, which comprises pharmaceutically acceptable salts or solvates of the desferriferrous pigment compound according to the technical scheme or the desferriferrous pigment compound prepared by the preparation method according to the technical scheme.
The invention provides a pharmaceutical composition, which comprises an effective active ingredient and pharmaceutically acceptable auxiliary materials; the effective active ingredients comprise the de-ferriferrous pigment compound prepared by the technical scheme, the de-ferriferrous pigment compound prepared by the preparation method of the technical scheme and one or more of the de-ferriferrous pigment compound derivatives of the technical scheme; the auxiliary materials are pharmaceutically or pharmaceutically acceptable auxiliary materials.
The invention provides application of the de-ferriferrous pigment compound, the de-ferriferrous pigment compound prepared by the preparation method of the technical scheme, the de-ferriferrous pigment compound derivative of the technical scheme or the pharmaceutical composition of the technical scheme in anti-tumor drugs.
Preferably, the tumor comprises one or more of colon adenocarcinoma tumor, liver cancer tumor, stomach cancer tumor, pancreatic cancer tumor and esophageal cancer tumor.
The invention provides a de-ferriferrous pigment compound which has a structure shown in a formula I. The iron-removing high-iron pigment compound provided by the invention belongs to an iron chelating agent compound, and the hydroxamic acid structure in the structure can be chelated with iron ions, so that the iron ion related metabolic pathway in cells is influenced, and the iron-removing high-iron pigment compound has better tumor cell growth inhibition activity and potential anti-tumor application prospect. As shown by the test results of the examples, the growth inhibition activities of HepG2 and Caco-2 cells of the iron-removing high-iron pigment compound for 72h provided by the invention are obviously better than that of positive control 5-fluorouracil.
The invention provides a preparation method of the de-iron high-iron pigment compound. The method takes the Morchella as the raw material, separates and purifies the Morchella for the first time to obtain the de-iron high-iron pigment compound with the structure shown in the formula I, has high safety and effectiveness of the de-iron high-iron pigment compound of Morchella sources, and greatly expands the pharmaceutical economic value of the Morchella; the preparation method is simple to operate, low in cost and low in cost, and raw materials are cheap and easy to obtain, so that the preparation method is suitable for industrial production; in addition, the preparation method provided by the invention can avoid using a large amount of organic raw materials, and is environment-friendly.
Drawings
FIG. 1 is a high performance liquid chromatogram of the iron-removing high-iron pigment compound prepared in example 1;
FIG. 2 is a graph showing the results of the activity of each of the compounds in test example 1 in inhibiting HepG2 growth;
FIG. 3 is a graph showing the results of the activity test of each compound in test example 1 in inhibiting Caco-2 growth.
Detailed Description
The invention provides a de-ferriferrous pigment compound which has a structure shown in a formula I:
in the formula I, R 1 、R 1 And R is 3 Independently selected from-OH, -CH 2 OH, hydrogen or C1-C6 alkyl, and R 1 、R 2 And R is 3 Are not hydrogen at the same time;
R 4 、R 5 and R is 6 Independently selected from-OH, -CH 2 OH, hydrogen or C1-C6 alkyl.
In the present invention, the R 1 ～R 6 The C1-C6 alkyl group in (2) is independently preferably methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, n-pentyl, 2-methylbutyl, 3-methylbutyl, 2-dimethylpropyl, n-hexyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2-dimethylbutyl, 2, 3-dimethylbutyl or 2, 4-dimethylbutyl.
The invention provides a preparation method of the de-iron high-iron pigment compound, which comprises the following steps:
(1) Mixing Morchella esculenta with the extractant, and extracting to obtain extract; the extracting agent is an organic solvent-water mixed solvent, and the organic solvent in the extracting solvent comprises one or more of alcohol solvents, nitrile solvents and ketone solvents;
(2) Carrying out sugar precipitation on the extract to obtain a sugar-removed crude extract;
(3) Separating the crude extract from the desugared extract by column chromatography to obtain a component Fr.2; the eluent adopted by the column chromatography separation is an organic solvent-water solvent, wherein the organic solvent comprises an alcohol solvent and/or a nitrile solvent, and the volume fraction of the organic solvent in the eluent is 0-100%;
(4) Subjecting the component Fr.2 to gel column chromatography separation to obtain a component Fr.2.2; the eluent for gel column chromatographic separation is an organic solvent-water mixed solvent, wherein the organic solvent comprises an alcohol solvent and/or a nitrile solvent, and the volume fraction of the organic solvent in the eluent is 30-60%;
(5) Separating the component Fr.2.2 by high performance liquid chromatography to obtain the de-iron high-iron pigment compound; the mobile phase adopted by the high performance liquid chromatography separation is an organic solvent-water mixed solvent, the organic solvent in the mobile phase comprises an alcohol solvent and/or a nitrile solvent, and the volume fraction of the organic solvent in the mobile phase is 4-20%.
In the present invention, all raw material components are commercially available products well known to those skilled in the art unless specified otherwise.
The Morchella esculenta and the extractant are mixed and extracted to obtain an extract.
In the invention, the extracting agent is an organic solvent-water mixed solvent, wherein the organic solvent in the extracting solvent comprises one or more of an alcohol solvent, a nitrile solvent and a ketone solvent, and preferably one or more of methanol, ethanol, acetonitrile and acetone; the volume fraction of the organic solvent in the extraction solvent is preferably 0 to 100%, more preferably 20 to 80%, and even more preferably 50 to 60%.
In the invention, the morchella is preferably dried morchella; the morchella is preferably crushed and then extracted; the drying method is not particularly limited, and a drying method well known to those skilled in the art may be adopted; the present invention is not particularly limited to the pulverization; in a specific embodiment of the invention, the comminution is preferably carried out in a multifunctional comminution mill.
In the present invention, the ratio of the dry weight (dry weight) of Morchella to the volume of the extractant (feed-liquid ratio) is preferably 1kg:2 to 20L, more preferably 1kg:3 to 10L, more preferably 1kg: 4-5L.
In the present invention, the extraction is preferably ultrasonic extraction, and the frequency of the ultrasonic extraction is preferably 30-50 kHz, more preferably 40kHz; the extraction temperature is preferably 20-50 ℃, more preferably 30-40 ℃; the number of times of the extraction is preferably 2 to 5 times, more preferably 2 to 3 times; the time of the single extraction is preferably 1 to 6 hours, more preferably 2 to 5 hours. The present invention is not particularly limited to the above-mentioned mixing, and the raw materials may be uniformly mixed, specifically, stirring and mixing. In a specific embodiment of the present invention, the ultrasonic extraction preferably includes sequentially performing a first ultrasonic extraction, a uniform stirring, and a second ultrasonic extraction, where the time of the first ultrasonic extraction and the second ultrasonic extraction are independently preferably 0.5 to 3 hours, and more preferably 1 to 2 hours; the first and second ultrasonic extractions are preferably performed in an ultrasonic cleaner. After each extraction is completed, the obtained extraction system is subjected to standing, the extracting solution and the sediment are respectively collected, the obtained sediment is subjected to next extraction, and the extracting solutions are combined.
After the extraction is completed, the present invention preferably concentrates the combined extracts to obtain an extract. The concentration of the present invention is not particularly limited, and may be performed by any concentration means known to those skilled in the art, such as evaporation to dryness.
After the extract is obtained, the invention carries out sugar precipitation on the extract to obtain a crude extract for removing sugar.
In the present invention, the solvent-sedimentation agent for sugar sedimentation preferably includes water and an organic solvent, and the organic solvent preferably includes an alcohol-based solvent and/or a nitrile-based solvent, more preferably includes one or more of ethanol, methanol, and acetonitrile; the volume ratio of the water to the organic solvent is preferably 1: (1.6 to 10), more preferably 1: (2 to 8), more preferably 1: (4-5). In the invention, the ratio of the dry weight of Morchella to the volume of the sedimentation agent is preferably 1kg:0.5 to 2.25L, more preferably 1kg: 0.75-1.5L. In a specific embodiment of the present invention, the sugar sedimentation is preferably: adding water into the extract, dispersing uniformly, adding organic solvent under stirring, standing, collecting supernatant, and concentrating to obtain crude extract. In the present invention, the temperature of the standing is preferably room temperature, the time of the standing is preferably 8 to 16 hours, more preferably 12 to 14 hours, and the purpose of the standing is to sufficiently settle the polysaccharide. The concentration of the present invention is not particularly limited, and may be performed by any concentration means known to those skilled in the art, such as evaporation to dryness.
After the crude extract of removing sugar is obtained, the crude extract of removing sugar is subjected to column chromatography separation to obtain a component Fr.2.
In the invention, the eluent adopted by the column chromatography separation is an organic solvent-water solvent, wherein the organic solvent comprises an alcohol solvent and/or a nitrile solvent, and more preferably one or more of methanol, ethanol and acetonitrile; the volume fraction of the organic solvent in the eluent is preferably 0 to 100%.
In the present invention, the above-mentioned crude desugared extract is preferably loaded by dissolving in water, and the amount of the water is not particularly limited in the present invention, and the crude desugared extract may be dissolved.
In the present invention, the column for the column chromatography separation is preferably an ODS-C18 column. In the invention, the elution mode of the column chromatography separation is preferably gradient elution, wherein the gradient elution is carried out by using organic solvent-water solvent with the volume fractions of 0 percent, 30 percent to 59 percent (preferably 40 percent to 50 percent), 60 percent to 90 percent (preferably 70 percent to 80 percent) and 100 percent in sequence, and the elution solutions are respectively collected and evaporated to dryness to obtain 4 components (one component is corresponding to each eluent), which are sequentially marked as a component Fr.1, a component Fr.2, a component Fr.3 and a component Fr.4.
After the component Fr.2 is obtained, the component Fr.2 is subjected to gel column chromatography separation to obtain the component Fr.2.2; the eluent for gel column chromatographic separation is an organic solvent-water mixed solvent, wherein the organic solvent comprises an alcohol solvent and/or a nitrile solvent, and preferably one or more of methanol, ethanol and acetonitrile; the volume fraction of the organic solvent in the eluent is 30-60%, preferably 40-50%. In the invention, 3 components are obtained by gel column chromatography separation and are sequentially marked as a component Fr.2.1, a component Fr.2.2 and a component Fr.2.3.
After the component Fr.2.2 is obtained, the component Fr.2.2 is subjected to high performance liquid chromatography to obtain the de-ferriferrous pigment compound; the mobile phase adopted by the high performance liquid chromatography separation is an organic solvent-water mixed solvent, and the organic solvent in the mobile phase comprises an alcohol solvent and/or a nitrile solvent, and more preferably one or more of methanol, ethanol and acetonitrile; the volume fraction of the organic solvent in the mobile phase is 4-20%, more preferably 6-10%; the high performance liquid chromatography separation is preferably preparative high performance liquid chromatography separation. In the present invention, the conditions for the high performance liquid chromatography separation include: the chromatographic column is preferably a C18 chromatographic column, and the flow rate of the mobile phase is preferably 10-20 mL/min, more preferably 15mL/min; the detection conditions are preferably UV 210nm.
The invention provides a derivative of a desferriferrous pigment compound, which comprises pharmaceutically acceptable salts or solvates of the desferriferrous pigment compound according to the technical scheme or the desferriferrous pigment compound prepared by the preparation method according to the technical scheme.
In the present invention, the pharmaceutically acceptable salt preferably includes sodium salt, potassium salt, ammonium salt or organic amine salt.
In the present invention, the pharmaceutically acceptable salt of the desferriferrous homoiron pigment compound is preferably obtained by reacting a desferriferrous homoiron pigment compound with an alkaline reagent. In the present invention, the alkaline agent preferably includes sodium hydroxide, potassium hydroxide, ammonia or an organic amine, and the present invention is not particularly limited, and organic amines well known to those skilled in the art may be used.
The solvate of the desferriferrous pigment compound is not particularly limited, and may be any solvate known to those skilled in the art.
The invention provides a pharmaceutical composition, which comprises an effective active ingredient and pharmaceutically acceptable auxiliary materials; the effective active ingredients comprise one or more of the de-ferriferrous pigment compounds prepared by the technical scheme, the de-ferriferrous pigment compounds prepared by the preparation method of the technical scheme and the de-ferriferrous pigment compound derivatives of the technical scheme; the auxiliary materials are pharmaceutically or pharmaceutically acceptable auxiliary materials. In the present invention, the auxiliary materials preferably include one or more of a carrier, an excipient and a diluent. The invention is not particularly limited to the food or pharmaceutical acceptable auxiliary materials, and the food or pharmaceutical acceptable auxiliary materials well known to those skilled in the art can be adopted.
The invention provides the application of the de-ferriferrous pigment compound, the de-ferriferrous pigment compound prepared by the preparation method of the technical scheme, the de-ferriferrous pigment compound derivative of the technical scheme or the pharmaceutical composition of the technical scheme in preparing antitumor drugs.
In the present invention, the tumor preferably includes one or more of colon adenocarcinoma tumor, liver cancer tumor, stomach cancer tumor, pancreatic cancer tumor and esophageal cancer tumor.
The technical solutions of the present invention will be clearly and completely described in the following in connection with the embodiments of the present invention. It will be apparent that the described embodiments are only some, but not all, embodiments of the invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Example 1
(1) Preparation of the Compounds
Collecting 2kg of Morchella (dry) sample, pulverizing with a multifunctional pulverizer, adding 8L of 50v/v% methanol water solution, stirring, placing into an ultrasonic cleaning instrument, ultrasonically extracting at room temperature and 40kHz for 1h, stirring again, continuing ultrasonic extraction for 1h, standing, respectively collecting the extract and precipitate, repeating the above extraction steps for 2 times, mixing the 3 times of extract, evaporating to dryness, adding 300mL of water into the obtained extract, fully dispersing, pouring into a 2L beaker, adding 1200mL of absolute ethyl alcohol under stirring, standing for 12h after adding to enable polysaccharide to fully settle, evaporating the collected supernatant to dryness to obtain the sugar-removing crude extract.
The crude extract after removing sugar is fully dissolved by water and then is subjected to ODS-C18 column chromatography, pure water, 50v/v% methanol aqueous solution, 80v/v% methanol aqueous solution and pure methanol are used for gradient elution, and eluent is respectively collected and evaporated to dryness, so that 4 components are obtained and are sequentially marked as component Fr.1, component Fr.2, component Fr.3 and component Fr.4. The fraction fr.2 was subjected to gel column chromatography (50 v/v% aqueous methanol solution) to obtain 3 fractions, which were sequentially designated as fraction fr.2.1, fraction fr.2.2 and fraction fr.2.3. Separating the component Fr.2.2 by preparative HPLC (mobile phase) to obtain 5 iron-removing high-iron pigment compounds, namely iron-removing bacteria, wherein the retention time is 24.73min; removing iron and high iron pigment, and keeping for 28.21min; compound MP-1 (R in formula I) 1 is-CH 2 OH), when retainedThe interval is 32.86min; removing iron and high iron pigment C, and keeping for 40.79min; compound MP-2 (R in formula I) 1 is-CH 3 ) The retention time is 48.69min, and the high performance liquid chromatogram of the above compound is shown in figure 1.
Wherein, the separation conditions of the preparative HPLC: c18 column (250X 10mm,5 μm) with mobile phase of 6v/v% acetonitrile water solution and mobile phase flow rate of 15mL/min; the detection conditions were UV 210nm.
(2) Structural characterization
Both compound MP-1 and compound MP-2 are white solids, are readily soluble in water, and UV absorption is terminal absorption. The molecular formula of the compound MP-1 is C 29 H 49 N 9 O 13 The molecular weight is 731.35. The molecular formula of the compound MP-2 is C 29 H 49 N 9 O 12 The molecular weight is 715.35. The molecular formula of the iron-removing high-iron pigment is C 29 H 49 N 9 O 12 Deferrioxacin has a molecular formula of C 29 H 49 N 9 O 12 The molecular formula of the de-ferriferrous pigment C is C 29 H 49 N 9 O 12 . The nuclear magnetic resonance identification data of each of the above compounds are shown in tables 1 to 2.
TABLE 1 Compounds from example 1 1 H-NMR data
TABLE 2 Compounds from example 1 13 C-NMR data
Test example 1
The 5 iron-removing high-iron pigments prepared in example 1 have activity of inhibiting tumor cell growth
Positive control: 5-fluorouracil.
The testing method comprises the following steps: the influence of the desferriferrous pigment monomer compound on Caco-2 and HepG2 cell proliferation is examined by applying a Sulfonyl Rhodamine B (SRB) detection method. Caco-2 and HepG2 cells were used at 10 before the experiment 4 Inoculating the density of individual cells/holes on a 96-hole micro-pore plate, taking deionized water as a negative control, taking 5-fluorouracil as a positive control, co-culturing the cells with the same batch of the compound to be detected with the concentration of 10 mu g/mL, and taking and culturing for 24 hours, 48 hours and 72 hours respectively; taking out 96-well microplates after finishing culturing, fixing cells in 5% trichloroacetic acid for 1h at 4 ℃, washing the cells with distilled water for 4 times, and dehydrating the microplates at room temperature; 100mL of 0.057% (wt/vol) aqueous SRB was added to each well and stained for 30 minutes, washed 4 times with 0.1% acetic acid, and then dehydrated again at room temperature; the stained cells were lysed in 100mL of 10mM Tris buffer and shaken on a horizontal shaker for 20 min to determine absorbance at 510nm using an enzyme-labeled instrument.
Cell viability= (a 1 -A 0 )/(A 2 -A 0 ) X 100%, where A 0 -zeroing the absorbance of the group; a is that 1 -absorbance of the sample group; a is that 2 Absorbance of negative control group.
The results of the activity test of each compound for inhibiting HepG2 growth are shown in FIG. 2 and Table 3, and the results of the activity test of each compound for inhibiting Caco-2 growth are shown in FIG. 3 and Table 3.
Average test results of the activity of the compounds of table 3 to inhibit growth of HepG2 and Caco-2 (n=6)
As can be seen from Table 3 and FIGS. 2 to 3, the HepG2 and Caco-2 cells prepared by the method for preparing the 5 iron-removing high-iron pigment compounds for 72h have obviously better growth inhibition activities than the positive control 5-fluorouracil.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Claims (5)
1. An iron-removing high-iron pigment compound is characterized by having the following structure:
or->。
2. The method for producing a desferriferrous iron pigment compound according to claim 1, comprising the steps of:
(1) Mixing Morchella esculenta with the extractant, and extracting to obtain extract; the extracting agent is water or an organic solvent-water mixed solvent, and the organic solvent in the extracting solvent comprises one or more of an alcohol solvent, a nitrile solvent and a ketone solvent; the ratio of the dry weight of Morchella to the volume of the extractant is 1kg: 2-20L; the times of extraction are 2-5 times, and the time of single extraction is 1-6 hours;
(2) Carrying out sugar precipitation on the extract to obtain a sugar-removed crude extract; the solvent sedimentation agent for sugar sedimentation comprises water and an organic solvent, wherein the volume ratio of the water to the organic solvent is 1: (1.6-10), wherein the organic solvent comprises an alcohol solvent and/or a nitrile solvent; the ratio of the dry weight of Morchella to the volume of the sedimentation agent is 1kg: 0.5-2.25L;
(3) Separating the crude extract from the desugared extract by column chromatography to obtain a component Fr.2; the eluent adopted by the column chromatography separation is an organic solvent-water solvent, wherein the organic solvent comprises an alcohol solvent and/or a nitrile solvent; the elution mode of the column chromatography separation is gradient elution, wherein the gradient elution is carried out by using organic solvent-water solvent with the volume fractions of 0%, 30-59%, 60-90% and 100% in sequence;
(4) Subjecting the component Fr.2 to gel column chromatography separation to obtain a component Fr.2.2; the eluent for gel column chromatographic separation is an organic solvent-water mixed solvent, wherein the organic solvent comprises an alcohol solvent and/or a nitrile solvent, and the volume fraction of the organic solvent in the eluent is 30-60%;
(5) Separating the component Fr.2.2 by high performance liquid chromatography to obtain the de-iron high-iron pigment compound; the mobile phase adopted by the high performance liquid chromatography separation is an organic solvent-water mixed solvent, the organic solvent in the mobile phase comprises an alcohol solvent and/or a nitrile solvent, and the volume fraction of the organic solvent in the mobile phase is 4-20%.
3. A desferriferrous homoiron pigment derivative comprising a pharmaceutically acceptable salt or solvate of a desferriferrous homoiron pigment compound as claimed in claim 1 or as prepared by a method as claimed in claim 2.
4. A pharmaceutical composition comprising an effective active ingredient and pharmaceutically acceptable excipients; the effective active ingredients comprise one or more of the de-ferriferrous pigment compounds of claim 1, the de-ferriferrous pigment compounds prepared by the preparation method of claim 2 and the de-ferriferrous pigment compound derivatives of claim 3; the auxiliary materials are pharmaceutically or pharmaceutically acceptable auxiliary materials.
5. Application of the de-ferriferrous pigment compound of claim 1, the de-ferriferrous pigment compound prepared by the preparation method of claim 2, the de-ferriferrous pigment compound derivative of claim 3 or the pharmaceutical composition of claim 4 in preparing antitumor drugs; the tumor comprises one or two of colon adenocarcinoma tumor and liver cancer tumor.
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| WO2015121704A2 (en) * | 2013-11-01 | 2015-08-20 | Miller Marvin J | Pathogen detection |
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| CN102516368A (en) * | 2011-12-21 | 2012-06-27 | 中国科学院南海海洋研究所 | Cyclopeptide-7 compounds and application thereof in preparation of anti-tumor medicines |
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| JP2019154282A (en) * | 2018-03-09 | 2019-09-19 | 月桂冠株式会社 | Alcoholic liquor, method for producing rice koji, method for producing alcoholic liquor, and unpleasant odor inhibiting composition |
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