CN117603053A - Diterpenoid compound with islet protection activity, and preparation method and application thereof - Google Patents
Diterpenoid compound with islet protection activity, and preparation method and application thereof Download PDFInfo
- Publication number
- CN117603053A CN117603053A CN202311633677.4A CN202311633677A CN117603053A CN 117603053 A CN117603053 A CN 117603053A CN 202311633677 A CN202311633677 A CN 202311633677A CN 117603053 A CN117603053 A CN 117603053A
- Authority
- CN
- China
- Prior art keywords
- compound
- petroleum ether
- islet
- ethyl acetate
- diterpenoid
- 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.)
- Pending
Links
- -1 Diterpenoid compound Chemical class 0.000 title claims abstract description 16
- 238000002360 preparation method Methods 0.000 title claims abstract description 10
- 230000000694 effects Effects 0.000 title abstract description 28
- 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 claims abstract description 36
- 239000008103 glucose Substances 0.000 claims abstract description 36
- 241000699670 Mus sp. Species 0.000 claims abstract description 26
- 230000006907 apoptotic process Effects 0.000 claims abstract description 20
- 230000006378 damage Effects 0.000 claims abstract description 9
- 230000001737 promoting effect Effects 0.000 claims abstract description 6
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 51
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims description 46
- 229940125904 compound 1 Drugs 0.000 claims description 42
- 239000003208 petroleum Substances 0.000 claims description 31
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 30
- IPCSVZSSVZVIGE-UHFFFAOYSA-N hexadecanoic acid Chemical compound CCCCCCCCCCCCCCCC(O)=O IPCSVZSSVZVIGE-UHFFFAOYSA-N 0.000 claims description 26
- ZSJLQEPLLKMAKR-GKHCUFPYSA-N streptozocin Chemical compound O=NN(C)C(=O)N[C@H]1[C@@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O ZSJLQEPLLKMAKR-GKHCUFPYSA-N 0.000 claims description 24
- ZSJLQEPLLKMAKR-UHFFFAOYSA-N Streptozotocin Natural products O=NN(C)C(=O)NC1C(O)OC(CO)C(O)C1O ZSJLQEPLLKMAKR-UHFFFAOYSA-N 0.000 claims description 23
- 229960001052 streptozocin Drugs 0.000 claims description 23
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 14
- 235000021314 Palmitic acid Nutrition 0.000 claims description 13
- 206010012601 diabetes mellitus Diseases 0.000 claims description 13
- WQEPLUUGTLDZJY-UHFFFAOYSA-N n-Pentadecanoic acid Natural products CCCCCCCCCCCCCCC(O)=O WQEPLUUGTLDZJY-UHFFFAOYSA-N 0.000 claims description 13
- 239000011259 mixed solution Substances 0.000 claims description 10
- 238000010898 silica gel chromatography Methods 0.000 claims description 9
- 238000004458 analytical method Methods 0.000 claims description 8
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 8
- 239000003480 eluent Substances 0.000 claims description 6
- 210000004153 islets of langerhan Anatomy 0.000 claims description 6
- 125000001033 ether group Chemical group 0.000 claims description 5
- HQVFCQRVQFYGRJ-UHFFFAOYSA-N formic acid;hydrate Chemical compound O.OC=O HQVFCQRVQFYGRJ-UHFFFAOYSA-N 0.000 claims description 5
- 238000000034 method Methods 0.000 claims description 5
- 239000000203 mixture Substances 0.000 claims description 5
- 238000001035 drying Methods 0.000 claims description 4
- 239000000284 extract Substances 0.000 claims description 4
- 238000010438 heat treatment Methods 0.000 claims description 4
- 210000005229 liver cell Anatomy 0.000 claims description 4
- 230000014759 maintenance of location Effects 0.000 claims description 4
- 238000010992 reflux Methods 0.000 claims description 4
- 239000002904 solvent Substances 0.000 claims description 4
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 3
- 238000000605 extraction Methods 0.000 claims description 3
- 210000003494 hepatocyte Anatomy 0.000 claims description 3
- AICOOMRHRUFYCM-ZRRPKQBOSA-N oxazine, 1 Chemical compound C([C@@H]1[C@H](C(C[C@]2(C)[C@@H]([C@H](C)N(C)C)[C@H](O)C[C@]21C)=O)CC1=CC2)C[C@H]1[C@@]1(C)[C@H]2N=C(C(C)C)OC1 AICOOMRHRUFYCM-ZRRPKQBOSA-N 0.000 claims description 3
- 239000000741 silica gel Substances 0.000 claims description 3
- 229910002027 silica gel Inorganic materials 0.000 claims description 3
- 238000009472 formulation Methods 0.000 claims description 2
- 238000004128 high performance liquid chromatography Methods 0.000 claims description 2
- 239000000825 pharmaceutical preparation Substances 0.000 claims description 2
- 238000002953 preparative HPLC Methods 0.000 claims description 2
- 238000004519 manufacturing process Methods 0.000 claims 1
- 210000004369 blood Anatomy 0.000 abstract description 24
- 239000008280 blood Substances 0.000 abstract description 24
- 239000003814 drug Substances 0.000 abstract description 13
- 238000002474 experimental method Methods 0.000 abstract description 12
- 150000001875 compounds Chemical class 0.000 abstract description 10
- 241000196324 Embryophyta Species 0.000 abstract description 6
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- 241001465754 Metazoa Species 0.000 abstract description 4
- 230000003914 insulin secretion Effects 0.000 abstract description 4
- 230000003908 liver function Effects 0.000 abstract description 4
- 230000000638 stimulation Effects 0.000 abstract description 3
- 244000025254 Cannabis sativa Species 0.000 abstract description 2
- 238000003782 apoptosis assay Methods 0.000 abstract description 2
- 235000021551 crystal sugar Nutrition 0.000 abstract description 2
- 229930002697 labdane diterpene Natural products 0.000 abstract description 2
- 210000004027 cell Anatomy 0.000 description 22
- NOESYZHRGYRDHS-UHFFFAOYSA-N insulin Chemical compound N1C(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(NC(=O)CN)C(C)CC)CSSCC(C(NC(CO)C(=O)NC(CC(C)C)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CCC(N)=O)C(=O)NC(CC(C)C)C(=O)NC(CCC(O)=O)C(=O)NC(CC(N)=O)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CSSCC(NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2C=CC(O)=CC=2)NC(=O)C(CC(C)C)NC(=O)C(C)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2NC=NC=2)NC(=O)C(CO)NC(=O)CNC2=O)C(=O)NCC(=O)NC(CCC(O)=O)C(=O)NC(CCCNC(N)=N)C(=O)NCC(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC(O)=CC=3)C(=O)NC(C(C)O)C(=O)N3C(CCC3)C(=O)NC(CCCCN)C(=O)NC(C)C(O)=O)C(=O)NC(CC(N)=O)C(O)=O)=O)NC(=O)C(C(C)CC)NC(=O)C(CO)NC(=O)C(C(C)O)NC(=O)C1CSSCC2NC(=O)C(CC(C)C)NC(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(N)CC=1C=CC=CC=1)C(C)C)CC1=CN=CN1 NOESYZHRGYRDHS-UHFFFAOYSA-N 0.000 description 14
- 210000002966 serum Anatomy 0.000 description 14
- 238000001514 detection method Methods 0.000 description 9
- 210000000496 pancreas Anatomy 0.000 description 9
- 108010003415 Aspartate Aminotransferases Proteins 0.000 description 8
- 102000004625 Aspartate Aminotransferases Human genes 0.000 description 8
- 102000004877 Insulin Human genes 0.000 description 7
- 108090001061 Insulin Proteins 0.000 description 7
- 229940125396 insulin Drugs 0.000 description 7
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 6
- 238000011160 research Methods 0.000 description 6
- 238000012360 testing method Methods 0.000 description 6
- 229930004069 diterpene Natural products 0.000 description 5
- 150000004141 diterpene derivatives Chemical class 0.000 description 5
- 238000004896 high resolution mass spectrometry Methods 0.000 description 5
- 150000003626 triacylglycerols Chemical class 0.000 description 5
- 108010028554 LDL Cholesterol Proteins 0.000 description 4
- 238000005481 NMR spectroscopy Methods 0.000 description 4
- 230000003833 cell viability Effects 0.000 description 4
- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 description 4
- 229940079593 drug Drugs 0.000 description 4
- 238000010828 elution Methods 0.000 description 4
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- 210000004923 pancreatic tissue Anatomy 0.000 description 4
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- 210000001519 tissue Anatomy 0.000 description 4
- 108010007622 LDL Lipoproteins Proteins 0.000 description 3
- 102000007330 LDL Lipoproteins Human genes 0.000 description 3
- 230000002424 anti-apoptotic effect Effects 0.000 description 3
- 238000004113 cell culture Methods 0.000 description 3
- 230000003247 decreasing effect Effects 0.000 description 3
- 238000011156 evaluation Methods 0.000 description 3
- 239000012091 fetal bovine serum Substances 0.000 description 3
- 238000000684 flow cytometry Methods 0.000 description 3
- 230000006872 improvement Effects 0.000 description 3
- 238000001543 one-way ANOVA Methods 0.000 description 3
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 description 3
- 239000001509 sodium citrate Substances 0.000 description 3
- 238000007619 statistical method Methods 0.000 description 3
- 238000001644 13C nuclear magnetic resonance spectroscopy Methods 0.000 description 2
- 238000005160 1H NMR spectroscopy Methods 0.000 description 2
- 102100036475 Alanine aminotransferase 1 Human genes 0.000 description 2
- 108010082126 Alanine transaminase Proteins 0.000 description 2
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 2
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 2
- 108010023302 HDL Cholesterol Proteins 0.000 description 2
- 108010010234 HDL Lipoproteins Proteins 0.000 description 2
- 102000015779 HDL Lipoproteins Human genes 0.000 description 2
- 241000699666 Mus <mouse, genus> Species 0.000 description 2
- LRHPLDYGYMQRHN-UHFFFAOYSA-N N-Butanol Chemical compound CCCCO LRHPLDYGYMQRHN-UHFFFAOYSA-N 0.000 description 2
- 229930182555 Penicillin Natural products 0.000 description 2
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 2
- 239000012980 RPMI-1640 medium Substances 0.000 description 2
- 108010087230 Sincalide Proteins 0.000 description 2
- 230000009471 action Effects 0.000 description 2
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- 230000003178 anti-diabetic effect Effects 0.000 description 2
- 239000003472 antidiabetic agent Substances 0.000 description 2
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- 238000010609 cell counting kit-8 assay Methods 0.000 description 2
- 235000012000 cholesterol Nutrition 0.000 description 2
- 238000012258 culturing Methods 0.000 description 2
- 238000007405 data analysis Methods 0.000 description 2
- SRCZQMGIVIYBBJ-UHFFFAOYSA-N ethoxyethane;ethyl acetate Chemical compound CCOCC.CCOC(C)=O SRCZQMGIVIYBBJ-UHFFFAOYSA-N 0.000 description 2
- DNJIEGIFACGWOD-UHFFFAOYSA-N ethyl mercaptane Natural products CCS DNJIEGIFACGWOD-UHFFFAOYSA-N 0.000 description 2
- 238000007446 glucose tolerance test Methods 0.000 description 2
- 230000012010 growth Effects 0.000 description 2
- 241000411851 herbal medicine Species 0.000 description 2
- 238000000338 in vitro Methods 0.000 description 2
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- 238000007912 intraperitoneal administration Methods 0.000 description 2
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- 150000002632 lipids Chemical class 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 230000007246 mechanism Effects 0.000 description 2
- XZWYZXLIPXDOLR-UHFFFAOYSA-N metformin Chemical group CN(C)C(=N)NC(N)=N XZWYZXLIPXDOLR-UHFFFAOYSA-N 0.000 description 2
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 description 2
- 238000000465 moulding Methods 0.000 description 2
- 230000007170 pathology Effects 0.000 description 2
- 229940049954 penicillin Drugs 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
- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 description 2
- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 description 2
- VTNULXUEOJMRKZ-UHFFFAOYSA-N 3-[4-(aminomethyl)-6-(trifluoromethyl)pyridin-2-yl]oxy-N-(2H-tetrazol-5-ylmethyl)benzamide Chemical compound N=1NN=NC=1CNC(C1=CC(=CC=C1)OC1=NC(=CC(=C1)CN)C(F)(F)F)=O VTNULXUEOJMRKZ-UHFFFAOYSA-N 0.000 description 1
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 description 1
- 108090000672 Annexin A5 Proteins 0.000 description 1
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- 238000008157 ELISA kit Methods 0.000 description 1
- 108010040476 FITC-annexin A5 Proteins 0.000 description 1
- 208000002705 Glucose Intolerance Diseases 0.000 description 1
- 241000202807 Glycyrrhiza Species 0.000 description 1
- 241000735432 Hydrastis canadensis Species 0.000 description 1
- 108010019160 Pancreatin Proteins 0.000 description 1
- 229930040373 Paraformaldehyde Natural products 0.000 description 1
- 241000013557 Plantaginaceae Species 0.000 description 1
- 241001529553 Scoparia <angiosperm> Species 0.000 description 1
- 244000265563 Scoparia dulcis Species 0.000 description 1
- 235000004500 Scoparia dulcis Nutrition 0.000 description 1
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 1
- ATJFFYVFTNAWJD-UHFFFAOYSA-N Tin Chemical compound [Sn] ATJFFYVFTNAWJD-UHFFFAOYSA-N 0.000 description 1
- ABRVLXLNVJHDRQ-UHFFFAOYSA-N [2-pyridin-3-yl-6-(trifluoromethyl)pyridin-4-yl]methanamine Chemical compound FC(C1=CC(=CC(=N1)C=1C=NC=CC=1)CN)(F)F ABRVLXLNVJHDRQ-UHFFFAOYSA-N 0.000 description 1
- 230000003187 abdominal effect Effects 0.000 description 1
- 230000003698 anagen phase Effects 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 210000000227 basophil cell of anterior lobe of hypophysis Anatomy 0.000 description 1
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- 231100000673 dose–response relationship Toxicity 0.000 description 1
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- 239000012259 ether extract Substances 0.000 description 1
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- 235000019253 formic acid Nutrition 0.000 description 1
- 230000006870 function Effects 0.000 description 1
- 238000003304 gavage Methods 0.000 description 1
- 230000002641 glycemic effect Effects 0.000 description 1
- 235000005679 goldenseal Nutrition 0.000 description 1
- 238000007490 hematoxylin and eosin (H&E) staining Methods 0.000 description 1
- 230000000055 hyoplipidemic effect Effects 0.000 description 1
- 238000005286 illumination Methods 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- LTINPJMVDKPJJI-UHFFFAOYSA-N iodinated glycerol Chemical compound CC(I)C1OCC(CO)O1 LTINPJMVDKPJJI-UHFFFAOYSA-N 0.000 description 1
- 230000001788 irregular Effects 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 230000037356 lipid metabolism Effects 0.000 description 1
- 238000004811 liquid chromatography Methods 0.000 description 1
- 238000013227 male C57BL/6J mice Methods 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
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- 210000000056 organ Anatomy 0.000 description 1
- 229940055695 pancreatin Drugs 0.000 description 1
- 239000012188 paraffin wax Substances 0.000 description 1
- 229920002866 paraformaldehyde Polymers 0.000 description 1
- 230000001575 pathological effect Effects 0.000 description 1
- 239000002243 precursor Substances 0.000 description 1
- 201000009104 prediabetes syndrome Diseases 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 238000004393 prognosis Methods 0.000 description 1
- 238000011894 semi-preparative HPLC Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 235000012424 soybean oil Nutrition 0.000 description 1
- 239000003549 soybean oil Substances 0.000 description 1
- 238000012306 spectroscopic technique Methods 0.000 description 1
- 239000012086 standard solution Substances 0.000 description 1
- 239000008223 sterile water Substances 0.000 description 1
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- UFTFJSFQGQCHQW-UHFFFAOYSA-N triformin Chemical compound O=COCC(OC=O)COC=O UFTFJSFQGQCHQW-UHFFFAOYSA-N 0.000 description 1
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- 235000015112 vegetable and seed oil Nutrition 0.000 description 1
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C69/00—Esters of carboxylic acids; Esters of carbonic or haloformic acids
- C07C69/76—Esters of carboxylic acids having a carboxyl group bound to a carbon atom of a six-membered aromatic ring
- C07C69/78—Benzoic acid esters
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/18—Drugs for disorders of the alimentary tract or the digestive system for pancreatic disorders, e.g. pancreatic enzymes
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
- A61P3/06—Antihyperlipidemics
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P5/00—Drugs for disorders of the endocrine system
- A61P5/48—Drugs for disorders of the endocrine system of the pancreatic hormones
- A61P5/50—Drugs for disorders of the endocrine system of the pancreatic hormones for increasing or potentiating the activity of insulin
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
- C07B2200/00—Indexing scheme relating to specific properties of organic compounds
- C07B2200/07—Optical isomers
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C2602/00—Systems containing two condensed rings
- C07C2602/02—Systems containing two condensed rings the rings having only two atoms in common
- C07C2602/14—All rings being cycloaliphatic
- C07C2602/26—All rings being cycloaliphatic the ring system containing ten carbon atoms
- C07C2602/28—Hydrogenated naphthalenes
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Life Sciences & Earth Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Veterinary Medicine (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Public Health (AREA)
- Pharmacology & Pharmacy (AREA)
- Engineering & Computer Science (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Diabetes (AREA)
- Endocrinology (AREA)
- Gastroenterology & Hepatology (AREA)
- Hematology (AREA)
- Obesity (AREA)
- Steroid Compounds (AREA)
Abstract
The invention belongs to the field of biological medicine, and in particular relates to a diterpenoid compound with islet protection activity, and a preparation method and application thereof. A novel labdane diterpene is isolated from whole plant of Crystal sugar grass and named as scoparicol E. In addition, the compounds were found to reduce PA-induced MIN6 apoptosis by anti-islet apoptosis assays performed on the compounds. Animal experiments prove that the compound can reduce the pancreatic weight and pancreatic index increase of mice caused by STZ stimulation; obviously reduces the fasting blood sugar of mice, obviously reduces glucose tolerance, has a certain lipid-lowering effect, has a certain protection effect on liver function, has an insulin secretion promoting effect, can improve the structural integrity of islets, and relieves the STZ-induced islet damage.
Description
Technical Field
The invention belongs to the field of biological medicine, and in particular relates to a diterpenoid compound with islet protection activity, and a preparation method and application thereof.
Background
The herb of rock candy (Scoparia dulcis l.) is a plant of the genus glycyrrhiza (Scoparia) of the family plantaginaceae, and is conventionally used for the prevention and treatment of diabetes. Through a great deal of literature researches, clinical and animal experiments prove that the rock candy herb has remarkable anti-diabetes efficacy, and the target organ of the rock candy herb is islet, but the efficacy components and the action mechanism of the rock candy herb for resisting diabetes are not clear through improving the islet. Therefore, the anti-diabetic diterpene based on islet protection is found from the Chinese herbal medicine, so that a scientific basis is provided for the research of the Chinese herbal medicine-based anti-diabetic medicine, and a theoretical basis and a research paradigm are provided for the research of the medicine based on the national medicine-based anti-islet beta cell apoptosis.
Disclosure of Invention
The technical scheme of the invention is realized as follows:
the first aspect of the invention provides a diterpenoid compound, which has a structural formula shown in a formula I:
the second aspect of the present invention provides a preparation method of the diterpenoid compound, comprising the following steps:
(1) Drying whole plant of herba Saussureae Involueratae, reflux extracting with 95% ethanol under heating, recovering ethanol, dissolving in water, and extracting with petroleum ether to obtain petroleum ether part;
(2) Separating the extract of herba Saussureae Involueratae petroleum ether extraction part by silica gel column chromatography, gradient eluting with petroleum ether/ethyl acetate, concentrating the eluate, recovering solvent, combining after TLC analysis and identification, and finally obtaining 12 fractions Fr.1-12;
(3) Separating Fr.10 by silica gel column chromatography, gradient eluting with petroleum ether/dichloromethane/ethyl acetate system, identifying and combining the eluates by TLC to finally obtain 11 subfractions Fr.10A-Fr.10K, wherein Fr.10H is separated and purified by semi-preparative high performance liquid chromatography to obtain the compound 1.
Further, in the step (2), silica gel used in the silica gel column chromatography is 200-300 meshes.
Further, in the step (2), the volume ratio of the petroleum ether and ethyl acetate mixed solution is 100:0-100:100; in the step (3), the volume ratio of the petroleum ether to the dichloromethane to the ethyl acetate mixed solution is 50:1:1-1:1:1.
Further, in the step (2), the volume ratio of the petroleum ether and ethyl acetate mixed solution is sequentially 100:0, 100:1, 100:2, 100:3, 100:4, 100:5, 100:8, 100:10, 100:12, 100:20, 100:50, 100:100; in the step (3), the volume ratio of the petroleum ether to the mixed solution of dichloromethane and ethyl acetate is sequentially 50:1:1, 30:1:1, 25:1:1, 20:1:1, 15:1:1, 12:1:1, 10:1:1, 8:1:1, 5:1:1 and 1:1:1.
Further, in the step (3), the high performance liquid chromatography column is YMC-pack ODS-Acolimn, and the eluent is: methanol-0.2% formic acid water=71: 29, flow rate of 2mL/min, retention time of compound 1 was 98 minutes.
The third aspect of the invention provides the use of the diterpenoid compound in the preparation of a pharmaceutical preparation for resisting islet cell apoptosis.
Further, in the application, the islet apoptosis is streptozotocin-induced islet damage and/or palmitic acid-induced apoptosis of MIN6 cells in diabetic mice.
In a fourth aspect, the invention provides the use of a diterpenoid compound as described above for the preparation of a formulation for promoting glucose consumption by hepatocytes.
Further, the liver cells are human liver cells HL-7702.
The invention has the beneficial effects that:
a novel labdane diterpene is isolated from whole plant of Crystal sugar grass and named as scoparicol E. Its structure is determined by nuclear magnetic resonance, HR-ESI-MS, and other spectroscopic techniques. In addition, the compounds were found to reduce PA-induced MIN6 apoptosis by anti-islet apoptosis assays performed on the compounds. Animal experiments prove that the compound can reduce the pancreatic weight and pancreatic index increase of mice caused by STZ stimulation when the administration dose is 5 mg/kg; obviously reduces the fasting blood sugar of mice, obviously reduces glucose tolerance, has a certain lipid-lowering effect, has a certain protection effect on liver function, has an insulin secretion promoting effect, can improve the structural integrity of islets, and relieves the STZ-induced islet damage.
Drawings
In order to more clearly illustrate the embodiments of the invention or the technical solutions of the prior art, the drawings which are used in the description of the embodiments or the prior art will be briefly described, it being obvious that the drawings in the description below are only some embodiments of the invention, and that other drawings can be obtained according to these drawings without inventive faculty for a person skilled in the art.
FIG. 1 Compound 1 1 H-NMR(600MHz,CDCl 3 )。
FIG. 2 Compound 1 13 C-NMR(150MHz,CDCl 3 )。
FIG. 3 effects of Compound 1 on PA-induced apoptosis of MIN6 cells. In comparison with the blank group, * p<0.05， ** p<0.005， *** p<0.001; in comparison with the control group of the model, # p<0.05, ## p<0.01, ### p<0.001。
FIG. 4 shows the results of the detection of anti-apoptotic effects of Compound 1 by Annexin-V/PI flow cytometry. .
FIG. 5 effect of Compound 1 on HL-7702cell glucose consumption. CK is control group, compound 1 is compound 1 treatment group with different concentrations, and MET is metformin group. In comparison with the control group, * p<0.05， ** p<0.005， *** p<0.001。
fig. 6. Effect of diterpene scoparicol E on abdominal and terminal blood glucose (n=10). (a) fasting blood glucose monitoring; (B) terminal blood glucose. And (3) injection: in comparison with the blank set of the cells, * p<0.05， ** p<0.005， *** p<0.001; in comparison with the set of models, # p<0.05。
fig. 7. Effect of diterpene scoparicol E on pancreas weight and pancreas index (n=10). (a) pancreatic weight; (B) pancreatic index. And (3) injection: in comparison with the blank set of the cells, * p<0.05， ** p<0.005，
*** p<0.001; in comparison with the set of models, # p<0.05, ## p<0.01, ### p<0.001。
the effect of scoparicol E on glucose tolerance by intraperitoneal injection (n=10).
FIG. 9 effect of diterpene scoparicol E on MLD-STZ induced pancreatic histopathology in diabetic mice (H & E, ×400).
FIG. 10. Influence of scoparicol E on serum biochemical index (A) Total Cholesterol (TC); (B) Total Triglycerides (TG); (C) glutamic oxaloacetic transaminase (AST); D. glutamic pyruvic transaminase (ALT); E. low Density Lipoprotein (LDL); F. high Density Lipoprotein (HDL), g.serum insulin levels. In comparison with the blank group, * p<0.05， ** p<0.005， *** p<0.001; in comparison with the control group of the model, # p<0.05, ## p<0.01, ### p<0.001。
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. 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.
1. Extraction and separation of compounds
Drying whole plant of herba Saussureae Involueratae, reflux extracting with 95% ethanol under heating, recovering ethanol, dissolving in water, and extracting with petroleum ether to obtain petroleum ether part; separating the extract of herba Saussureae Involueratae petroleum ether extract by silica gel column chromatography (200-300 mesh), gradient eluting with petroleum ether/ethyl acetate (100:0-100:100), concentrating the eluate, recovering solvent, and combining after TLC analysis and identification to obtain 12 fractions (Fr.1-12).
Fr.10 is separated by silica gel column chromatography, gradient elution is carried out by a petroleum ether/dichloromethane/ethyl acetate system (50:1:1-1:1:1), eluent is identified and combined by TLC, and finally 11 subfractions (Fr.10A-Fr.10K) are obtained, wherein Fr.10H is separated and purified by semi-preparative HPLC to obtain a compound 1, and the volume ratio of eluent is 71:29 methanol-0.2% formic acid water, retention time of compound 1 was 98min, mass 95.2mg. The specific operation steps are as follows:
the dried whole plant (50.0 kg) of the rock candy herb is extracted by heating and refluxing with 95% ethanol twice for 2 hours each time. Recovering solvent, dissolving with water, sequentially extracting with petroleum ether, dichloromethane and n-butanol to obtain petroleum ether part 350g, subjecting petroleum ether part to silica gel (200-300 mesh) column chromatography, and respectively adopting petroleum ether-ethyl acetate 100:0-100:100 Gradient elution) to obtain 12 fractions, wherein the volume ratio of petroleum ether and ethyl acetate mixed solution is sequentially 100:0 (Fr.1), 100:1 (Fr.2), 100:2 (Fr.3), 100:3 (Fr.4), 100:4 (Fr.5), 100:5 (Fr.6), 100:8 (Fr.7), 100:10 (Fr.8), 100:12 (Fr.9), 100:20 (Fr.10), 100:50 (Fr.11) and 100:100 (Fr.12). Collecting eluent with petroleum ether-ethyl acetate ratio of 100:20 (Fr.10), concentrating under reduced pressure, and drying to obtain extract.
Wherein the fraction 10 (Fr.10) is subjected to silica gel column chromatography, petroleum ether is adopted to carry out gradient elution, the volume ratio of dichloromethane to ethyl acetate (the volume ratio is 50:1:1-1:1:1), the volume ratio of petroleum ether to mixed solution of dichloromethane and ethyl acetate is sequentially 50:1:1 (Fr.10.1), 30:1:1 (Fr.10.2), 25:1:1 (Fr.10.3), 20:1:1 (Fr.10.4), 15:1:1 (Fr.10.5), 12:1:1 (Fr.10.6), 10:1:1 (Fr.10.7), 8:1:1 (Fr.10.8), 5:1:1 (Fr.10.9), and 1:1 (Fr.10.10), wherein the Fr.10.8 is subjected to semi-preparative high-efficiency liquid chromatography to carry out elution to obtain the compound 1. The HPC column was YMC-pack ODS-A column (250 mm. Times.10 mm,5 μm,12nm, YMC Co., td., kyoto, japan) and the eluent was 71 by volume: 29 methanol-0.2% formic acid water, wherein 0.2% formic acid water is 0.2% formic acid aqueous solution by volume ratio, flow rate is 2mL/min, and retention time of compound 1 is 98 minutes.
2. Structure identification of Compounds
2.1 their structure was determined by Nuclear Magnetic Resonance (NMR), high resolution mass spectrometry (HR-ESI-MS) and the like. The specific data are shown in Table 1 and FIGS. 1-2.
Table 1 NMR data for compound 1
Wherein, 1 the H-NMR test condition was 600MHz; 13 C-NMR test conditions were 150MHz, CDCl 3 。
Table 2 high resolution mass spectrometry data for compounds 1 and 2
| Sample name | Mass spectrometer | Ionization mode | Actual measurement value | Theoretical value | Molecular formula |
| Compound 1 | HR-ESI-MS | [M+Na] + | 463.2456 | 463.2455 | C 27 H 36 O 5 |
The structure of compound 1 was finally determined as follows:
compound 1 is a novel diterpenoid compound, which is named as scoparicol E according to structure.
2. Activity detection of Compound 1 on MIN6 islet cell (viability) protection at different concentrations
2.1 cell culture
MIN6 cells at 37deg.C, 5% CO 2 MIN6 cells were cultured in RPMI-1640 medium containing 10% fetal bovine serum, 50. Mu.M mercaptoethanol and 1% penicillin. Taking MIN6 cells in logarithmic growth phase, inoculating the MIN6 cells into a 96-well plate, culturing for 24 hours, and adding the medicine to be tested.
2.2 evaluation of Compound 1 Activity against islet apoptosis
Cell treatment: MIN6 cells with good growth state are uniformly inoculated into a 96-well plate, RPMI-1640 medium (10% FBS, 50. Mu.M mercaptoethanol, 10% P/S) 100. Mu.L/well is added at 1X 105cells/mL, and the 96-well plate is placed at 37℃and 5% CO 2 Culturing in an incubator for 24 hours. Discarding the original culture medium, and performing no treatment on the control group (CK); adding a drug-containing culture medium into an orifice plate, and respectively adding compound 1 with different concentrations into toxicity detection experiment groups; anti-islet cell apoptosis experimental model group was only added with PA (300 μm), compound 1 group was given 300 μm Palmitic Acid (PA), while different concentrations of compound 1 to be screened (scoparicol E) were given for 24h. Each set of 5 concentration gradients was replicated in parallel wells and the experiment was independently repeated 3 times.
Cell viability was determined by CCK-8 method:
after MIN6 cells were subjected to the above conditioning intervention, CCK-8 solution was added to 96-well plates at 100. Mu.L per well using cell-free medium as a blank. Then put into a furnace at 37℃,5％CO 2 After incubation for 1h, the Optical Density (OD) was finally measured at a wavelength of 450nm using a microplate reader. And cell viability was calculated.
Cell viability (%) = (treatment OD-blank OD)/(control OD-blank OD) ×100%2.3 data analysis:
statistical analysis
Experimental data are expressed as mean ± standard deviation (mean ± SD), with One-wayANOVA analysis in GraphPad Prism 8.0 software, p <0.05 indicates statistical differences, p <0.01 indicates significant differences, and p <0.001 indicates very significant differences.
2.4 evaluation results of Compound 1 Activity against islet apoptosis
The results are shown in FIG. 3. FIG. 3A shows the toxicity test results of compound 1 on MIN6 cells, showing that the cell activity can be kept normal within the concentration range of 0.195-12.5 mu M, and no significant difference is caused between the compound 1 and the control group. Cell activity decreased significantly (p < 0.001) after increasing the concentration to 25 μm, and cells were not viable at a concentration of 50 μm. The concentration of the anti-apoptotic activity test compound 1 was then set in the range of 0.195-12.5. Mu.M.
Compared with the control group, the model group is added with PA, so that the cell activity is obviously reduced, and the success of modeling is indicated. In contrast, compound 1 (0.39, 0.78, 1.56, 3.125, 6.25, 12.5 μm) at different concentrations significantly increased cell viability (76.13 ± 7.119,84.12 ± 1.216,93.69 ± 4.610,100.2 ± 2.348,107.0 ±4.474and 107.1±2.401%, p < 0.001) and increased dose-dependent after 24h of intervention treatment compared to model group PA (300 μm). Compound 1 was suggested to be effective in improving PA-induced MIN6 apoptosis (fig. 3B).
2.5Annexin-V/PI flow cytometry detection of Compound 1 anti-apoptotic results
MIN6 cells treated in "evaluation of anti-islet cell apoptosis activity of Compound 1" were individually taken and the effect of Compound 1 (scoparicol E) on PA-induced MIN6 apoptosis was examined using Annexin V-FITC flow cytometry. The percentage of PA-induced MIN6 apoptosis (22.86%) was significantly increased compared to control cells (8.44%). Compound 1 group (6.25 μm) showed a significant decrease in MIN6 apoptosis (13.26%) (fig. 4). Thus, compound 1 at a concentration of 6.25 μm can reduce PA-induced MIN6 apoptosis.
3. Investigation of the Effect of Compound 1 on the sugar consumption of HL-7702cells at different concentrations 3.1 cell culture
Human hepatocyte cell line HL-7702cells were cultured in DMEM (Dulbecco's Modified Eagle's Medium) containing 10% heat-inactivated fetal bovine serum and 1% penicillin/streptomycin at a concentration of 80% -90%,37℃at a temperature of 5% CO 2 Is cultured in a cell culture incubator.
3.2 in vitro sugar consumption experiments
(1) HL-7702cells with good growth state are resuspended into single cells after pancreatin digestion, and uniformly inoculated into 96-well plates with 100 μl per well. When the cells grow to 80% -90% density, the administration treatment is carried out, the original culture medium is discarded, and the drug-containing culture medium is added into an orifice plate to be set as a control group (CK), a compound 1 different concentration treatment (compound 1) and a metformin group (Met). 5 multiple holes are respectively arranged, and the culture is carried out for 24 hours in an incubator.
(2) Taking out the culture plate from the incubator, preparing a new 96-well plate, making corresponding marks, adding 2 mu L of Kong Daice samples subjected to drug administration and glucose standard solution into the 96-well plate, adding 200 mu L/well of glucose consumption detection working solution preheated at 37 ℃, uniformly mixing on a 96-well plate oscillator, placing the mixture into the incubator at 37 ℃ for incubation for 10min, taking out the well plate, and detecting OD values of all the wells in an enzyme-labeling instrument at 505nm wavelength.
(3) And (3) calculating:
glucose consumption concentration = initial glucose concentration- (initial glucose concentration x test drug OD value/standard OD value).
Standard culture (initial glucose concentration) =25.1 mM
(4) Data analysis:
statistical analysis
Experimental data are expressed as mean ± standard deviation (mean ± SD), with One-wayANOVA analysis in GraphPad Prism 8.0 software, p <0.05 indicates statistical differences, p <0.01 indicates significant differences, and p <0.001 indicates very significant differences.
(5) In vitro results of sugar consumption experiments
The results are shown in FIG. 5. Compared with the CK group, the glucose consumption of HL-7702cells is improved after intervention treatment of compound 1 (0.39, 0.78, 1.56, 3.125, 6.25 and 12.5 mu M) with different concentrations, and different dosage improvement degrees are different. The concentration of the compound 1 is 0.39 and 3.125 mu M, and the improvement is remarkable * p<0.05 A) is provided; the concentration is 0.78, 1.56, 6.25 and 12.5 mu M, the lifting is more obvious ** p<0.005)。
4. Protection effect of compound 1 on multiple low-dose streptozotocin-induced islet damage in diabetic mice
4.1 preparation of Streptozotocin (STZ) solution
Precisely weighing 20mg of STZ, placing into a 15mL sterile centrifuge tube, and wrapping with tinfoil to avoid illumination. The mixture is placed at the temperature of minus 20 ℃ for preservation, taken out when in use, placed on ice, and added with 5mL of sodium citrate buffer solution before injection, so that the mixture is completely dissolved and uniformly mixed. The injection was completed within 15 min.
4.2 administration of Compound 1
Compound 1 (scoparicol E) was administered at a low dose of 5mg/kg (scoparicol E-L) and at a high dose of 10mg/kg (scoparicol E-H). After the medicaments with different concentrations are prepared, the medicaments are stored at 4 ℃ for standby.
4.3 grouping and modeling of animals
After basal feed-adaptive feeding for 7d, 40 male C57BL/6J mice of 4 weeks of age were randomly divided into blank groups (Normal), streptozotocin model group (STZ), low dose group (scoparicol E-L), and high dose group (scoparicol E-H). Each group of 10. Mice were fasted for 12 hours (no water for fasted: 21:00-the next day 9:00), after which the corresponding doses of drug were infused for intervention: the blank and model groups were given vegetable oil (in experiments using goldenseal soybean oil); the scoparicol E treatment group was perfused once daily at a concentration of 5mg/kg at a low dose and 10mg/kg at a high dose for 7 consecutive days. From the beginning of the first lavage, 1h after each lavage, the model group and the dosing group were intraperitoneally injected with 50mg/kg STZ (dissolved in sodium citrate buffer pH 4.5), and the blank group was intraperitoneally injected with the same volume of sodium citrate buffer for 5 consecutive days. All mice were then fed with basal feed, blood was collected on day 21, serum samples were taken for further testing, and tissue samples were collected for further analysis.
4.4 Experimental methods
4.4.1 routine detection
Body weight was measured twice weekly.
4.4.2 blood glucose determination
Fasting blood glucose was measured on days 1,7, 14, and 21, and all mice were fasted and not watered for 8 hours on day 21, and blood was taken after fasting blood glucose measurement.
4.4.3 determination of pancreatic weight
The pancreas was removed and placed on ice and the pancreas weight of each group of mice was measured.
4.4.4 pancreatic histopathological detection
Mice were sacrificed and dissected, pancreatic tissue was rapidly removed, cleaned with normal saline, appropriate sized tissue was cut, the pancreas was fixed in 10% paraformaldehyde tissue fixative for 48h to allow adequate fixation of the tissue, after conventional paraffin embedding sections (5 μm), stained with hematoxylin-eosin (HE) and photographed under light.
4.4.5 intraperitoneal glucose tolerance test (IPGTT)
On day 20, all experimental animals were fasted for 6 hours, at which time the measured fasting blood glucose was taken as a value at time point 0. 4.00g glucose was accurately weighed and dissolved in 20mL of sterile water to prepare a 20% glucose solution, which was administered to all experimental animals by intraperitoneal injection, with a glucose intake of 2g/kg. Blood glucose values were measured 30, 60, 120, 180min after glucose intake, respectively, and then plotted at time points. All glycemic tests of the present invention used a blood glucose meter (Roche, ACCU-CHEK) to detect blood glucose levels.
4.4.6 serum Biochemical index analysis
After standing for 4 hours at room temperature, centrifuging at 4 ℃ at 3500rpm for 15 minutes, transferring the supernatant to a new centrifuge tube, and detecting the levels of Total Cholesterol (TC), triglyceride (TG), high-density lipoprotein cholesterol (HDL-c), low-density lipoprotein cholesterol (LDL-c), glutamic-pyruvic transaminase (ALT) and aspartate transaminase (AST) in the serum of the mouse according to the instruction of the kit. Serum Insulin levels were detected using an instrin enzyme-linked immunosorbent assay kit.
4.4.9 statistical analysis
Experimental data are expressed as mean ± standard deviation (mean ± SD), with One-wayANOVA analysis in GraphPad Prism 8.0 software, p <0.05 indicates statistical differences, p <0.01 indicates significant differences, and p <0.001 indicates very significant differences.
4.5 experimental results
4.5.1 effects on blood glucose
As can be seen from fig. 6A, after 7 days of administration of compound 1 (scoparicol E) (5 mg/kg,10 mg/kg) by gavage, the observation was continued for 14 days, and the fasting blood glucose was significantly elevated in the model group compared to the blank group, and the results showed that: the blood glucose levels of the fasting blood in the scoparicol E (5 mg/kg) low dose group were significantly reduced compared to the model group (STZ-treated group). Figure 6B shows a significant decrease in fasting blood glucose levels in the low dose dosing group compared to the model group at day 21 # p<0.05)。
4.5.2 pancreas weight and pancreas index
Figure 7 shows that STZ model group had an elevated pancreatic index compared to the blank group. Compared with the model group, the pancreas index of the low dose group of the compound 1 is reduced, and other indexes have no obvious difference. The STZ model group mice weighed 0.1591 ± 0.02572g, and pancreas index 0.007578 ± 0.001433g, respectively, significantly higher than the blank group mice (fig. 7). Following compound 1 (5 mg/kg) intervention, the pancreatic weight and pancreatic index of mice were significantly reduced to 0.09773 ± 0.01081g and 0.005521 ± 0.0008448, respectively (fig. 7).
4.5.3 intraperitoneal glucose tolerance test (IPGTT)
Compound 1 low dose group (scoparicol E-L) (5 mg/kg) reduced IPGTT following intervention therapy (fig. 8), indicating that scoparicol E was able to improve STZ-induced impaired glucose tolerance in diabetic mice.
4.5.4 effects on pancreatic tissue pathology
The pancreatic tissue HE staining of each group (figure 9) shows that the islets of mice in the blank group are obvious, normal in size, round in shape, normal in cell arrangement and free from pathological phenomena; in the STZ model group, the shape of the islets of the mice is irregular, the boundary is blurred, the number of cells in the islets is reduced, and the vacuoles are obviously increased. And after the scoparicol E is interfered, the integrity of the islet structure is obviously improved. Thus, scoparicol E treatment can ameliorate STZ-induced islet damage in mice.
4.5.5 serum Biochemical index detection
Results show (see fig. 10): serum TC and LDL-C levels were 3.609 + -0.2054, 1.509 + -0.1098 mM, and 3.950 + -0.4220 mM, respectively, and serum lipid levels were higher in STZ-model mice than in normal mice (p < 0.05) (FIGS. 10A, E, F). Following treatment with scoparimol E (5 mg/kg), serum lipid levels of TC and LDL-C were reduced to 2.796 + -0.4296,1.305 + -0.1485 mM (FIGS. 10A, E). These results indicate that scoparicol E can improve diabetes-related lipid metabolism. Serum ALT and AST levels reached 47.88+ -4.368,45.96 + -9.084U/L in mice from STZ model group, which was significantly higher than in the blank group (ALT, 34.95 + -2.704U/L, AST,42.97 + -2.959U/L). serum ALT and AST levels were reduced to 36.51 + -6.364,25.98 + -6.978U/L, respectively, following scoparicol E (5 mg/kg) treatment, well below STZ model group mice (FIGS. 10C, D). Insulin levels (3.04.+ -. 0.10 IU/L) were significantly lower in the STZ model group than in the blank group (3.16.+ -. 0.054 IU/L), and insulin levels increased to 3.18.+ -. 0.04IU/L following treatment with scoparimol E (5 mg/kg) (FIG. 10G).
The model group (STZ treated group) had elevated TC and TG values compared to the blank group, while the scoparicol E administered group had reduced TC, TG and LDL values compared to the model group, with some hypolipidemic effect. When the AST and ALT values of the model group (STZ treatment group) were increased compared with those of the blank group, the administered scoparicol E group was decreased compared with those of the model group, and the liver function was protected to some extent. Compared with the blank group, the insulin level of the model group is obviously reduced, and then the insulin level of the administered scoparicol E (5 mg/kg) is obviously increased compared with that of the model group, so that the model group has the function of promoting insulin secretion.
The study adopts an MLD-STZ induced diabetes mouse islet damage model, and scoparicol E carries out in-vivo islet protection efficacy experiments and explores the action mechanism thereof. The experiment uses 50mg/kg STZ to carry out multiple small-dose molding, and the fasting blood glucose value is detected after 5 days, and when the fasting blood glucose value is higher than 11.1mmol/L, the molding is considered to be successful. StZ-induced diabetic mice were found to have increased fasting blood glucose with a significant decrease in serum insulin levels due to islet cell injury death. Administration of scoparimol E (5 mg/kg) in dry prognosis can alleviate the significant decrease in fasting blood glucose in mice caused by STZ stimulation, while the low dose group of scoparimol E (5 mg/kg) has significantly decreased glucose tolerance. The serum biochemical index detection result shows that the scoparicol E has a certain lipid-lowering effect, has a certain protection effect on liver function, and has an insulin secretion promoting effect. In addition, the pancreatic tissue pathology observation shows that the scoparicol E can improve the integrity of islet structures, relieve STZ-induced islet damage and the like.
In conclusion, the rock candy herb diterpene scoparicol E can obviously reduce the blood sugar level of the MLD-STZ induced diabetic mice when the oral dosage is 5mg/kg, and can improve islet damage. The experiment provides a research foundation for developing the precursor compound for resisting islet cell apoptosis and provides a research basis for developing the oral medicament for treating diabetes.
The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, alternatives, and improvements that fall within the spirit and scope of the invention.
Claims (10)
1. The diterpenoid compound is characterized by having a structural formula shown in a formula I:
2. the method for preparing diterpenoid compounds according to claim 1, comprising the following steps:
(1) Drying whole plant of herba Saussureae Involueratae, reflux extracting with 95% ethanol under heating, recovering ethanol, dissolving in water, and extracting with petroleum ether to obtain petroleum ether part;
(2) Separating the extract of herba Saussureae Involueratae petroleum ether extraction part by silica gel column chromatography, gradient eluting with petroleum ether/ethyl acetate, concentrating the eluate, recovering solvent, combining after TLC analysis and identification, and finally obtaining 12 fractions Fr.1-12;
(3) Separating Fr.10 by silica gel column chromatography, gradient eluting with petroleum ether/dichloromethane/ethyl acetate system, identifying and combining the eluates by TLC to finally obtain 11 subfractions Fr.10A-Fr.10K, wherein Fr.10H is separated and purified by semi-preparative high performance liquid chromatography to obtain the compound 1.
3. The method for producing diterpenoid compounds according to claim 2, wherein in the step (2), silica gel used for silica gel column chromatography is 200 to 300 mesh.
4. The method for preparing diterpenoid compounds according to claim 2, wherein in the step (2), the volume ratio of petroleum ether and ethyl acetate mixed solution is 100:0-100:100; in the step (3), the volume ratio of the petroleum ether to the dichloromethane to the ethyl acetate mixed solution is 50:1:1-1:1:1.
5. The method for preparing diterpenoid compounds according to claim 2, wherein in the step (2), the volume ratio of the petroleum ether and ethyl acetate mixed solution is sequentially 100:0, 100:1, 100:2, 100:3, 100:4, 100:5, 100:8, 100:10, 100:12, 100:20, 100:50, 100:100; in the step (3), the volume ratio of the petroleum ether to the mixed solution of dichloromethane and ethyl acetate is sequentially 50:1:1, 30:1:1, 25:1:1, 20:1:1, 15:1:1, 12:1:1, 10:1:1, 8:1:1, 5:1:1 and 1:1:1.
6. The method of claim 2, wherein in the step (3), the high performance liquid chromatography column is YMC-pack ODS-se:Sup>A column, and the eluent is: methanol-0.2% formic acid water=71: 29, flow rate of 2mL/min, retention time of compound 1 was 98 minutes.
7. Use of diterpenoid compounds according to claim 1 for the preparation of pharmaceutical preparations against islet cell apoptosis.
8. The use according to claim 7, wherein: in the application, the islet cell apoptosis is streptozotocin-induced islet damage and/or palmitic acid-induced MIN6 cell apoptosis of diabetic mice.
9. Use of a diterpenoid compound as defined in claim 1 for the preparation of a formulation for promoting glucose consumption by hepatocytes.
10. The use according to claim 9, wherein: the liver cells are human liver cells HL-7702.
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