CN117244001A - Application of bromhidrosis root petroleum ether extract in preparation of medicines for treating non-small cell lung cancer - Google Patents
Application of bromhidrosis root petroleum ether extract in preparation of medicines for treating non-small cell lung cancer Download PDFInfo
- Publication number
- CN117244001A CN117244001A CN202311218021.6A CN202311218021A CN117244001A CN 117244001 A CN117244001 A CN 117244001A CN 202311218021 A CN202311218021 A CN 202311218021A CN 117244001 A CN117244001 A CN 117244001A
- Authority
- CN
- China
- Prior art keywords
- petroleum ether
- lung cancer
- root
- small cell
- cell lung
- 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
- 239000003208 petroleum Substances 0.000 title claims abstract description 103
- 239000012259 ether extract Substances 0.000 title claims abstract description 83
- 208000035985 Body Odor Diseases 0.000 title claims abstract description 65
- 206010055000 Bromhidrosis Diseases 0.000 title claims abstract description 63
- 208000002154 non-small cell lung carcinoma Diseases 0.000 title claims abstract description 31
- 208000029729 tumor suppressor gene on chromosome 11 Diseases 0.000 title claims abstract description 31
- 239000003814 drug Substances 0.000 title claims abstract description 16
- 229940079593 drug Drugs 0.000 title description 5
- 238000002360 preparation method Methods 0.000 title description 3
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims abstract description 40
- 230000006907 apoptotic process Effects 0.000 claims abstract description 26
- 230000002438 mitochondrial effect Effects 0.000 claims abstract description 14
- 241000219764 Dolichos Species 0.000 claims abstract description 13
- 230000018199 S phase Effects 0.000 claims abstract description 10
- 230000035755 proliferation Effects 0.000 claims abstract description 8
- 239000002904 solvent Substances 0.000 claims abstract description 8
- 230000001404 mediated effect Effects 0.000 claims abstract description 4
- 230000022131 cell cycle Effects 0.000 claims description 10
- 239000007788 liquid Substances 0.000 claims description 3
- 238000002156 mixing Methods 0.000 claims description 3
- 206010027476 Metastases Diseases 0.000 claims description 2
- 239000000463 material Substances 0.000 claims description 2
- 230000009401 metastasis Effects 0.000 claims description 2
- 238000000034 method Methods 0.000 claims description 2
- 239000002994 raw material Substances 0.000 claims description 2
- 239000003795 chemical substances by application Substances 0.000 claims 8
- 239000002775 capsule Substances 0.000 claims 1
- 239000003937 drug carrier Substances 0.000 claims 1
- 239000000839 emulsion Substances 0.000 claims 1
- 238000001914 filtration Methods 0.000 claims 1
- 239000002674 ointment Substances 0.000 claims 1
- 239000006187 pill Substances 0.000 claims 1
- 238000010298 pulverizing process Methods 0.000 claims 1
- 239000000829 suppository Substances 0.000 claims 1
- 230000005012 migration Effects 0.000 abstract description 8
- 238000013508 migration Methods 0.000 abstract description 8
- 230000009545 invasion Effects 0.000 abstract description 7
- 230000001939 inductive effect Effects 0.000 abstract description 4
- 230000000259 anti-tumor effect Effects 0.000 abstract description 2
- 230000002401 inhibitory effect Effects 0.000 abstract description 2
- 239000002246 antineoplastic agent Substances 0.000 abstract 1
- 229940041181 antineoplastic drug Drugs 0.000 abstract 1
- 230000004071 biological effect Effects 0.000 abstract 1
- 210000004027 cell Anatomy 0.000 description 101
- 230000000694 effects Effects 0.000 description 20
- 239000000284 extract Substances 0.000 description 17
- 239000000243 solution Substances 0.000 description 14
- 238000010186 staining Methods 0.000 description 12
- 239000000126 substance Substances 0.000 description 12
- 230000005764 inhibitory process Effects 0.000 description 10
- 239000001963 growth medium Substances 0.000 description 9
- -1 humulone epoxide Chemical class 0.000 description 9
- 210000001700 mitochondrial membrane Anatomy 0.000 description 9
- 206010028980 Neoplasm Diseases 0.000 description 8
- NVEQFIOZRFFVFW-RGCMKSIDSA-N caryophyllene oxide Chemical compound C=C1CC[C@H]2O[C@]2(C)CC[C@H]2C(C)(C)C[C@@H]21 NVEQFIOZRFFVFW-RGCMKSIDSA-N 0.000 description 8
- 231100000673 dose–response relationship Toxicity 0.000 description 8
- 239000000975 dye Substances 0.000 description 8
- 239000000203 mixture Substances 0.000 description 8
- 229920000776 Poly(Adenosine diphosphate-ribose) polymerase Polymers 0.000 description 7
- 230000003828 downregulation Effects 0.000 description 7
- 230000000144 pharmacologic effect Effects 0.000 description 7
- 102000003952 Caspase 3 Human genes 0.000 description 6
- 108090000397 Caspase 3 Proteins 0.000 description 6
- 102000004039 Caspase-9 Human genes 0.000 description 6
- 108090000566 Caspase-9 Proteins 0.000 description 6
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 6
- 102000055102 bcl-2-Associated X Human genes 0.000 description 6
- 108700000707 bcl-2-Associated X Proteins 0.000 description 6
- 230000015572 biosynthetic process Effects 0.000 description 6
- 238000002474 experimental method Methods 0.000 description 6
- 230000014759 maintenance of location Effects 0.000 description 6
- 102000004169 proteins and genes Human genes 0.000 description 6
- 108090000623 proteins and genes Proteins 0.000 description 6
- 102100026802 72 kDa type IV collagenase Human genes 0.000 description 5
- 101710151806 72 kDa type IV collagenase Proteins 0.000 description 5
- 102100021569 Apoptosis regulator Bcl-2 Human genes 0.000 description 5
- 241001655728 Celastrus Species 0.000 description 5
- 235000017186 Celastrus paniculatus Nutrition 0.000 description 5
- 241000219051 Fagopyrum Species 0.000 description 5
- 235000009419 Fagopyrum esculentum Nutrition 0.000 description 5
- 101000971171 Homo sapiens Apoptosis regulator Bcl-2 Proteins 0.000 description 5
- 239000012980 RPMI-1640 medium Substances 0.000 description 5
- 238000012258 culturing Methods 0.000 description 5
- 239000002609 medium Substances 0.000 description 5
- 230000001105 regulatory effect Effects 0.000 description 5
- GRWFGVWFFZKLTI-IUCAKERBSA-N (-)-α-pinene Chemical compound CC1=CC[C@@H]2C(C)(C)[C@H]1C2 GRWFGVWFFZKLTI-IUCAKERBSA-N 0.000 description 4
- AKTLQTMAOYZJGI-UHFFFAOYSA-N 3-Eicosene Natural products CCCCCCCCCCCCCCCCC=CCC AKTLQTMAOYZJGI-UHFFFAOYSA-N 0.000 description 4
- NVEQFIOZRFFVFW-UHFFFAOYSA-N 9-epi-beta-caryophyllene oxide Natural products C=C1CCC2OC2(C)CCC2C(C)(C)CC21 NVEQFIOZRFFVFW-UHFFFAOYSA-N 0.000 description 4
- 206010058467 Lung neoplasm malignant Diseases 0.000 description 4
- 238000004458 analytical method Methods 0.000 description 4
- 230000005775 apoptotic pathway Effects 0.000 description 4
- 238000004820 blood count Methods 0.000 description 4
- RSYBQKUNBFFNDO-UHFFFAOYSA-N caryophyllene oxide Natural products CC1(C)CC2C(=C)CCC3OC3(C)CCC12C RSYBQKUNBFFNDO-UHFFFAOYSA-N 0.000 description 4
- 230000003013 cytotoxicity Effects 0.000 description 4
- 231100000135 cytotoxicity Toxicity 0.000 description 4
- CBFCDTFDPHXCNY-UHFFFAOYSA-N icosane Chemical compound CCCCCCCCCCCCCCCCCCCC CBFCDTFDPHXCNY-UHFFFAOYSA-N 0.000 description 4
- 201000005202 lung cancer Diseases 0.000 description 4
- 208000020816 lung neoplasm Diseases 0.000 description 4
- 210000003470 mitochondria Anatomy 0.000 description 4
- 230000000877 morphologic effect Effects 0.000 description 4
- INAAIJLSXJJHOZ-UHFFFAOYSA-N pibenzimol Chemical compound C1CN(C)CCN1C1=CC=C(N=C(N2)C=3C=C4NC(=NC4=CC=3)C=3C=CC(O)=CC=3)C2=C1 INAAIJLSXJJHOZ-UHFFFAOYSA-N 0.000 description 4
- 238000012546 transfer Methods 0.000 description 4
- 230000003827 upregulation Effects 0.000 description 4
- AKTLQTMAOYZJGI-FNORWQNLSA-N (3E)-3-Icosene Chemical compound CCCCCCCCCCCCCCCC\C=C\CC AKTLQTMAOYZJGI-FNORWQNLSA-N 0.000 description 3
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 3
- 240000000425 Chaenomeles speciosa Species 0.000 description 3
- 235000005078 Chaenomeles speciosa Nutrition 0.000 description 3
- 241000196324 Embryophyta Species 0.000 description 3
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 241000174389 Premna puberula Species 0.000 description 3
- 201000011510 cancer Diseases 0.000 description 3
- 150000001875 compounds Chemical class 0.000 description 3
- 238000001514 detection method Methods 0.000 description 3
- 239000012091 fetal bovine serum Substances 0.000 description 3
- 238000000684 flow cytometry Methods 0.000 description 3
- 238000002290 gas chromatography-mass spectrometry Methods 0.000 description 3
- 239000000523 sample Substances 0.000 description 3
- 239000012192 staining solution Substances 0.000 description 3
- 238000005406 washing Methods 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- JXRRJAPYKWLLHV-KFRFFZJSSA-N (-)-Humulene epoxide II Natural products C[C@]12O[C@@H]1CC/C=C(/C)\CC(C)(C)/C=C/C2 JXRRJAPYKWLLHV-KFRFFZJSSA-N 0.000 description 2
- QTGAEXCCAPTGLB-UOAUIWSESA-N (1r,4e,8e,11r)-4,7,7,11-tetramethyl-12-oxabicyclo[9.1.0]dodeca-4,8-diene Chemical compound C1CC(/C)=C/CC(C)(C)\C=C\C[C@@]2(C)O[C@@H]21 QTGAEXCCAPTGLB-UOAUIWSESA-N 0.000 description 2
- SPURMHFLEKVAAS-UHFFFAOYSA-N 1-docosene Chemical compound CCCCCCCCCCCCCCCCCCCCC=C SPURMHFLEKVAAS-UHFFFAOYSA-N 0.000 description 2
- NNZRVXTXKISCGS-UHFFFAOYSA-N 1-methoxy-2-propan-2-ylbenzene Chemical compound COC1=CC=CC=C1C(C)C NNZRVXTXKISCGS-UHFFFAOYSA-N 0.000 description 2
- 102000000905 Cadherin Human genes 0.000 description 2
- 108050007957 Cadherin Proteins 0.000 description 2
- 241001256368 Clematis chinensis Species 0.000 description 2
- RMFGNMMNUZWCRZ-UHFFFAOYSA-N Humulone Natural products CC(C)CC(=O)C1=C(O)C(O)(CC=C(C)C)C(O)=C(CC=C(C)C)C1=O RMFGNMMNUZWCRZ-UHFFFAOYSA-N 0.000 description 2
- LRHPLDYGYMQRHN-UHFFFAOYSA-N N-Butanol Chemical compound CCCCO LRHPLDYGYMQRHN-UHFFFAOYSA-N 0.000 description 2
- 108050000637 N-cadherin Proteins 0.000 description 2
- 229930040373 Paraformaldehyde Natural products 0.000 description 2
- 241000131771 Premna Species 0.000 description 2
- 206010040904 Skin odour abnormal Diseases 0.000 description 2
- 102000004142 Trypsin Human genes 0.000 description 2
- 108090000631 Trypsin Proteins 0.000 description 2
- 241001073567 Verbenaceae Species 0.000 description 2
- 230000003213 activating effect Effects 0.000 description 2
- 230000004913 activation Effects 0.000 description 2
- MVNCAPSFBDBCGF-UHFFFAOYSA-N alpha-pinene Natural products CC1=CCC23C1CC2C3(C)C MVNCAPSFBDBCGF-UHFFFAOYSA-N 0.000 description 2
- 230000001640 apoptogenic effect Effects 0.000 description 2
- 235000013527 bean curd Nutrition 0.000 description 2
- 230000000903 blocking effect Effects 0.000 description 2
- 239000012159 carrier gas Substances 0.000 description 2
- 230000004663 cell proliferation Effects 0.000 description 2
- 239000006285 cell suspension Substances 0.000 description 2
- 238000010367 cloning Methods 0.000 description 2
- 230000001472 cytotoxic effect Effects 0.000 description 2
- 238000007865 diluting Methods 0.000 description 2
- 239000012153 distilled water Substances 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 239000012894 fetal calf serum Substances 0.000 description 2
- 239000000834 fixative Substances 0.000 description 2
- QTGAEXCCAPTGLB-UHFFFAOYSA-N humulene epoxide II Natural products C1CC(C)=CCC(C)(C)C=CCC2(C)OC21 QTGAEXCCAPTGLB-UHFFFAOYSA-N 0.000 description 2
- 210000003734 kidney Anatomy 0.000 description 2
- 238000004949 mass spectrometry Methods 0.000 description 2
- 239000011159 matrix material Substances 0.000 description 2
- 229920002866 paraformaldehyde Polymers 0.000 description 2
- YKNWIILGEFFOPE-UHFFFAOYSA-N pentacosane Chemical compound CCCCCCCCCCCCCCCCCCCCCCCCC YKNWIILGEFFOPE-UHFFFAOYSA-N 0.000 description 2
- 229920000642 polymer Polymers 0.000 description 2
- 239000010453 quartz Substances 0.000 description 2
- GRWFGVWFFZKLTI-UHFFFAOYSA-N rac-alpha-Pinene Natural products CC1=CCC2C(C)(C)C1C2 GRWFGVWFFZKLTI-UHFFFAOYSA-N 0.000 description 2
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N silicon dioxide Inorganic materials O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 2
- 239000002356 single layer Substances 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
- 239000006228 supernatant Substances 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 239000012588 trypsin Substances 0.000 description 2
- 230000029663 wound healing Effects 0.000 description 2
- YXHLJMWYDTXDHS-IRFLANFNSA-N 7-aminoactinomycin D Chemical compound C[C@H]1OC(=O)[C@H](C(C)C)N(C)C(=O)CN(C)C(=O)[C@@H]2CCCN2C(=O)[C@@H](C(C)C)NC(=O)[C@H]1NC(=O)C1=C(N)C(=O)C(C)=C2OC(C(C)=C(N)C=C3C(=O)N[C@@H]4C(=O)N[C@@H](C(N5CCC[C@H]5C(=O)N(C)CC(=O)N(C)[C@@H](C(C)C)C(=O)O[C@@H]4C)=O)C(C)C)=C3N=C21 YXHLJMWYDTXDHS-IRFLANFNSA-N 0.000 description 1
- 108700012813 7-aminoactinomycin D Proteins 0.000 description 1
- 102000000412 Annexin Human genes 0.000 description 1
- 108050008874 Annexin Proteins 0.000 description 1
- 102000011727 Caspases Human genes 0.000 description 1
- 108010076667 Caspases Proteins 0.000 description 1
- 238000002965 ELISA Methods 0.000 description 1
- 230000010190 G1 phase Effects 0.000 description 1
- 230000010337 G2 phase Effects 0.000 description 1
- 108010043121 Green Fluorescent Proteins Proteins 0.000 description 1
- 101000911513 Homo sapiens Uncharacterized protein FAM215A Proteins 0.000 description 1
- 208000019255 Menstrual disease Diseases 0.000 description 1
- 208000037093 Menstruation Disturbances Diseases 0.000 description 1
- 206010027339 Menstruation irregular Diseases 0.000 description 1
- 206010029155 Nephropathy toxic Diseases 0.000 description 1
- 206010029350 Neurotoxicity Diseases 0.000 description 1
- 206010030113 Oedema Diseases 0.000 description 1
- 108010019160 Pancreatin Proteins 0.000 description 1
- 229930182555 Penicillin Natural products 0.000 description 1
- 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 1
- 206010034464 Periarthritis Diseases 0.000 description 1
- 201000002661 Spondylitis Diseases 0.000 description 1
- 208000021945 Tendon injury Diseases 0.000 description 1
- 206010044221 Toxic encephalopathy Diseases 0.000 description 1
- 102100026728 Uncharacterized protein FAM215A Human genes 0.000 description 1
- 239000004480 active ingredient Substances 0.000 description 1
- 238000010598 annexinV-PE /7AAD assay Methods 0.000 description 1
- 230000001093 anti-cancer Effects 0.000 description 1
- 238000007664 blowing Methods 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 239000007795 chemical reaction product Substances 0.000 description 1
- 238000002512 chemotherapy Methods 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
- 238000003776 cleavage reaction Methods 0.000 description 1
- 230000005757 colony formation Effects 0.000 description 1
- 230000000295 complement effect Effects 0.000 description 1
- 238000005336 cracking Methods 0.000 description 1
- 239000013078 crystal Substances 0.000 description 1
- 238000003235 crystal violet staining Methods 0.000 description 1
- 239000012228 culture supernatant Substances 0.000 description 1
- 210000000805 cytoplasm Anatomy 0.000 description 1
- 230000034994 death Effects 0.000 description 1
- 231100000517 death Toxicity 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 230000001627 detrimental effect Effects 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 230000002222 downregulating effect Effects 0.000 description 1
- 238000001647 drug administration Methods 0.000 description 1
- 230000005284 excitation Effects 0.000 description 1
- 239000004744 fabric Substances 0.000 description 1
- 210000002950 fibroblast Anatomy 0.000 description 1
- 239000007850 fluorescent dye Substances 0.000 description 1
- 238000009650 gentamicin protection assay Methods 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 description 1
- 230000001969 hypertrophic effect Effects 0.000 description 1
- 230000000415 inactivating effect Effects 0.000 description 1
- 230000002779 inactivation Effects 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 230000006882 induction of apoptosis Effects 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 231100001231 less toxic Toxicity 0.000 description 1
- 231100000053 low toxicity Toxicity 0.000 description 1
- 210000004072 lung Anatomy 0.000 description 1
- 230000036210 malignancy Effects 0.000 description 1
- 230000003211 malignant effect Effects 0.000 description 1
- 210000001161 mammalian embryo Anatomy 0.000 description 1
- 238000001819 mass spectrum Methods 0.000 description 1
- 239000002398 materia medica Substances 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- LCGLNKUTAGEVQW-UHFFFAOYSA-N methyl monoether Natural products COC LCGLNKUTAGEVQW-UHFFFAOYSA-N 0.000 description 1
- 230000009456 molecular mechanism Effects 0.000 description 1
- 239000000178 monomer Substances 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 230000007694 nephrotoxicity Effects 0.000 description 1
- 231100000417 nephrotoxicity Toxicity 0.000 description 1
- 230000007135 neurotoxicity Effects 0.000 description 1
- 231100000228 neurotoxicity Toxicity 0.000 description 1
- 238000010606 normalization Methods 0.000 description 1
- PIRWNASAJNPKHT-SHZATDIYSA-N pamp Chemical compound C([C@@H](C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](C)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCCNC(N)=N)C(N)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CO)NC(=O)[C@H](C)NC(=O)[C@@H](NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](C)N)C(C)C)C1=CC=CC=C1 PIRWNASAJNPKHT-SHZATDIYSA-N 0.000 description 1
- 229940055695 pancreatin Drugs 0.000 description 1
- 230000037361 pathway Effects 0.000 description 1
- 235000010987 pectin Nutrition 0.000 description 1
- 229920001277 pectin Polymers 0.000 description 1
- 239000001814 pectin Substances 0.000 description 1
- 229940049954 penicillin Drugs 0.000 description 1
- 230000035699 permeability Effects 0.000 description 1
- 230000008823 permeabilization Effects 0.000 description 1
- 239000013641 positive control Substances 0.000 description 1
- 238000011085 pressure filtration Methods 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 238000001959 radiotherapy Methods 0.000 description 1
- 238000010992 reflux Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- 238000007789 sealing Methods 0.000 description 1
- 239000012679 serum free medium Substances 0.000 description 1
- 238000010008 shearing Methods 0.000 description 1
- 229960005322 streptomycin Drugs 0.000 description 1
- 238000001356 surgical procedure Methods 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 208000004371 toothache Diseases 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 238000010877 transwell invasion assay Methods 0.000 description 1
- 238000009834 vaporization Methods 0.000 description 1
- 230000008016 vaporization Effects 0.000 description 1
- 238000001262 western blot Methods 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/85—Verbenaceae (Verbena family)
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
- A61P35/04—Antineoplastic agents specific for metastasis
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/30—Extraction of the material
- A61K2236/35—Extraction with lipophilic solvents, e.g. Hexane or petrol ether
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/50—Methods involving additional extraction steps
- A61K2236/51—Concentration or drying of the extract, e.g. Lyophilisation, freeze-drying or spray-drying
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/50—Methods involving additional extraction steps
- A61K2236/53—Liquid-solid separation, e.g. centrifugation, sedimentation or crystallization
Landscapes
- Health & Medical Sciences (AREA)
- Natural Medicines & Medicinal Plants (AREA)
- Life Sciences & Earth Sciences (AREA)
- Pharmacology & Pharmacy (AREA)
- Chemical & Material Sciences (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- General Health & Medical Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- Medicinal Chemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Medical Informatics (AREA)
- Botany (AREA)
- Biotechnology (AREA)
- Alternative & Traditional Medicine (AREA)
- Engineering & Computer Science (AREA)
- Mycology (AREA)
- Epidemiology (AREA)
- Microbiology (AREA)
- Oncology (AREA)
- Medicines Containing Plant Substances (AREA)
Abstract
The invention discloses application of a bromhidrosis root petroleum ether extract in preparing a medicine for treating non-small cell lung cancer. The invention discloses a petroleum ether extract of falcate dolichos root for treating non-small cell lung cancer, which is prepared by extracting falcate dolichos root with solvent petroleum ether, inhibits proliferation of non-small cell lung cancer cells through S phase retardation, can exert anti-tumor biological effect by inducing mitochondrial mediated apoptosis and inhibiting migration and invasion, and has the prospect of developing new anti-tumor drugs for treating non-small cell lung cancer and the like.
Description
Technical Field
The invention belongs to the field of biological medicine, and in particular relates to application of a bromhidrosis root petroleum ether extract in preparation of a medicine for treating non-small cell lung cancer.
Background
Lung Cancer is one of the common cancers, accounting for approximately 18% of the total deaths from Cancer worldwide (CA Cancer J Clin,2020, 71:209-249). The incidence of chinese lung cancer is 73.3 cases per hundred thousand, with 27.21% of male cancers and 21.92% of female cancers being the highest mortality cancers among the first of the malignancies (Evid Based Complement Alternat Med,2018, 2018:8567905). Of the total incidence of lung cancer, non-small-cell lung cancer (NSCLC) is the highest, accounting for about 80-85% (Biomark Res,2023, 11:7). Chemotherapy has been used for lung cancer treatment in combination with surgery and radiation therapy at present, but has serious side effects such as nephrotoxicity and neurotoxicity, which are detrimental to patient survival (Mol Cell Biochem,2021,476 (1): 57-68). Therefore, there is an urgent need to develop effective drugs with less toxic side effects.
Bromhidrosis (Premna puberula pamp.) is an edible and medicinal plant of Premna genus (Premna) of Verbenaceae (Verbenaceae), and is distributed in Guizhou, guangxi, yunnan, sichuan, hunan, hubei, guangxi, guangdong, gansu and Fujian (Chinese plant Zhi [ M ]. Beijing: beijing scientific press, 1994, volume 17: 1-49). The leaves of the bromhidrosis are widely used for making cool bean curd (commonly called magical bean curd) due to the rich pectin content (mountain agricultural biology report, 2011,30 (1): 82-86;J Appl Pharmaceut Sci,2012,2 (9): 031-035). As a traditional Chinese medicine, the bromhidrosis root and stem have the effects of tonifying kidney yang and dispelling wind-damp, and can be used for treating kidney deficiency, hypertrophic spondylitis, scapulohumeral periarthritis and irregular menstruation; the leaves can be used for treating menoxenia, edema, malignant boil, burn, tendon injury and fracture; in addition, it can be used for treating toothache (Chinese plant Shi M. Beijing: beijing science publication, 1994,1994, volume 17: 1-49; chinese herbal Shi M, 1999, volume 6: 587-589;Common Chinese Materia Medica,Singapore:Springer,2022,pp:440-441). The root of bromhidrosis is always used as a traditional Chinese medicine, but the chemical components and pharmacological activity of the root of bromhidrosis are not researched, and no related report of anticancer effect of petroleum ether extract of the root is found.
Disclosure of Invention
The purpose of the invention is that: the application of the bromhidrosis root petroleum ether extract in preparing the medicine for treating the non-small cell lung cancer is provided, the new application of the bromhidrosis root petroleum ether extract is developed, and a new choice is provided for the medicine for treating the non-small cell lung cancer.
The invention also discovers the inhibition effect of the bromhidrosis root petroleum ether extract on the proliferation of human non-small cell lung cancer cells, the cell cycle blocking effect, the apoptosis induction and the transfer inhibition effect, and identifies the chemical components of the bromhidrosis root petroleum ether extract.
The technical scheme adopted by the invention is as follows:
the bromhidrosis root petroleum ether extract is prepared by the following method:
fresh root of bromhidrosis (500 g) was triturated and charged into a round bottom flask. Sequentially reflux-extracting with petroleum ether, ethyl acetate, n-butanol and water as solvents. In the case of extraction with different solvents, solvent (2L) was used for reflux extraction for 2h, repeated twice. The four extracts collected were filtered, concentrated under reduced pressure in a rotary evaporator to remove the solvent, and then the rest was freeze-dried except for the petroleum ether extract. Samples of the four extracts were kept in closed brown glass bottles and placed in a desiccator.
The chemical composition of the extract of falcate root petroleum ether was determined by GC-MS (see table 1 in detail), wherein the main chemical compositions were Luo Songfen (ferroginol, 15.0%), (+) -cycloalfalfa (12.0%), humulone epoxide II (humulene epoxide II, 7.6%), eicosane (5.2%), α -pinene (α -copane, 5.0%), caryophyllene oxide (caryophyllene oxide, 4.4%), o-isopropylphenyl methyl ether (o-isopropylanise, 4.3%), 1-docodiene (1-docosine, 4.3%), 7-isopropyl-1,1,4A-trimethyl-2, 3,4,9 (dehyduabietan, 4.2%), and 3-eicosene (3-eicosen, 3.9%).
The invention researches the cytotoxic activity of the bromhidrosis root petroleum ether extract on the human non-small cell lung cancer by experiments, and takes cisplatin as a positive control. The result shows that the compound has lower cytotoxicity to normal cells and obvious inhibition effect on the proliferation of human non-small cell lung cancer cells; has promoting effect on apoptosis of human non-small cell lung cancer cells; has blocking effect on human non-small cell lung cancer cell cycle; has inhibiting effect on metastasis of human non-small cell lung cancer cells.
By adopting the technical scheme, the invention discovers the new application of the bromhidrosis root petroleum ether extract in the aspect of treating the non-small cell lung cancer for the first time, provides a new choice for the medicine for treating the human non-small cell lung cancer, and has important application value in the pharmaceutical industry.
Drawings
FIG. 1 shows the cytotoxicity comparison of the extract of Chaenomeles speciosa (L.) of the petroleum ether against non-small cell lung cancer (A549) and normal cells (MRC-5);
FIG. 2 shows the effect of bromhidrosis root petroleum ether extract on A549 cell clone formation;
FIG. 3 is the effect of bromhidrosis root petroleum ether extract on A549 cell cycle. (A) After the bromhidrosis root petroleum ether extract is treated, the detection result of an A549 cell cycle flow cytometry is counted in proportion of (B) A549 cells in different periods (G1 and S, G2);
FIG. 4 is a morphological observation of apoptosis of A549 cells by the extract of Fagopyrum armpit odor petroleum ether. (A) morphological influence of the bromhidrosis root petroleum ether extract on A549 cells, (B) analysis of apoptosis of the A549 cells induced by the bromhidrosis root petroleum ether extract by AO/EB staining, and (C) analysis of apoptosis of the A549 cells induced by the bromhidrosis root petroleum ether extract by Hoechst 33258 staining;
FIG. 5 is a flow cytometer analysis of apoptosis induced by the extract of Fagophus body odor root Petroleum ether in A549 cells. (A) A549 cells are treated with different concentrations of bromhidrosis root petroleum ether extract for 24 hours, stained with Annexin V-PE and 7-AAD, analyzed by a flow cytometer, and (B) statistical results of total apoptotic cells and living cell proportion are obtained;
FIG. 6 is a graph showing the effect of JC-1 staining analysis of the petroleum ether extract of Fagopyrum bromhidrosis on the mitochondrial membrane potential of A549 cells;
FIG. 7 shows the effect of the petroleum ether extract of Chaenomeles speciosa on the protein associated with the mitochondrial apoptosis pathway of A549 cells. (A, C) expression levels of apoptosis-related proteins total Cytc, mitochondrial Cytc, bax, bcl-2, pro-caspase 9, clear-caspase 9, pro-caspase 3, clear-caspase 3 and clear-PARP protein levels after 48h treatment of the petroleum ether extract of Fox bromhidrosis, (B, D, E) total Cytc, mitochondrial Cytc, bax, bcl-2, pro-caspase 9, clear-caspase 9, pro-caspase 3, clear-caspase 3 and clear-PARP band gray scale quantitative statistics;
FIG. 8 shows the effect of the petroleum ether extract of Chaenomeles speciosa on migration and invasion of A549 cells and the expression of related proteins. (A, B) detecting migration ability of the pessary petroleum ether extract to a549 cells with a wound healing test and quantitatively analyzing the migration ability with mobility (%); (C, D) Transwell invasion assay to determine invasiveness and quantitatively analyze invasiveness; (E, F) WB detection of the expression of transfer related proteins (MMP-2, N-cadherin) in A549 cells after treatment of the bromhidrosis root petroleum ether extract, and quantitative statistics of the gray values of the bands of the MMP-2 and N-cadherin.
Detailed Description
Embodiments of the invention: fresh faggots (harvested place: guizhou Guiyang; identified by the professor Hu Guoxiong of Guizhou), crushed raw materials and petroleum ether are mixed according to a feed-liquid ratio of 1:4, the mixed materials are extracted for 4 hours in an extractor, the extracting solution is collected, reduced pressure filtration and concentration are carried out to remove the solvent, and the faggots petroleum ether extract is obtained and is stored in a refrigerator at 4 ℃ in a sealing way.
The chemical components of the bromhidrosis root petroleum ether extract in the embodiment are as follows: the chemical composition of the petroleum ether extract of the falcate dolichos root is identified by GC-MS. GC-MS analysis conditions: sample injection amount is 2 mu L, the chromatographic column is an HP-5MS (60 m multiplied by 0.25mm multiplied by 0.25 mu m) elastic quartz capillary column, the initial temperature is 70 ℃ (reserved for 2 min), the temperature is increased to 180 ℃ (55 min) at 2 ℃/min, the temperature is increased to 310 ℃ (13 min and reserved for 14 min) at 10 ℃/min, and the operation time is as follows: 84min; the temperature of the vaporization chamber is 250 ℃; the carrier gas is high purity He (99.999%); 18.53psi pre-column pressure, 1.0mL/min carrier gas flow, split ratio 20:1, solvent delay time: 6.0min; the ion source is an EI source; the ion source temperature is 230 ℃; the temperature of the quadrupole rods is 150 ℃; electron energy 70eV; emission current 34.6 μa; multiplier voltage 1847V; the interface temperature is 280 ℃; the mass range is 29-500 amu. And (3) searching and checking Nist2020 and Wiley275 standard mass spectrograms of each peak in the total ion flow chart through a mass spectrum computer data system, determining chemical components, and determining the relative mass fraction of each chemical component by using a peak area normalization method. The chemical composition of the petroleum ether extract of falcate root is shown in table 1, and 50 chemical compositions were identified, accounting for 98.6% of the total peak area, wherein the main chemical compositions were Luo Songfen (ferroginol, 15.0%), (+) -cycloalfalfa (cyclodrive, 12.0%), humulone epoxide II (humulene epoxide II, 7.6%), eicosane (pentacosane, 5.2%), α -pinene (α -copane, 5.0%), caryophyllene oxide (caryophyllene oxide, 4.4%), o-isopropyl dimethyl ether (o-isopropylanisole, 4.3%), 1-docodiene (1-docosene, 4.3%), 7-isopropyl-1,1,4A-trimethyl-2, 3,4,9 (deoabane, 4.2%), and 3-eicosene (3-eicosene, 3.9%).
Table 1 chemical composition of the petroleum ether extract of falcate dolichos root
a calculating Retention Index (RI): warp C 8 -C 30 HP-5MS (60 m. Times.0.25 mm. Times.0.25 μm) retention index on elastic quartz capillary column calculated for n-alkane mixing;
b database Retention Index (RI): retention index from NIST2020 mass spectrometry database;
note that: the active ingredient was identified based on Wiley275 and NIST2020 mass spectrometry databases and comparison of calculated retention index with database retention index.
Pharmacological example 1: cytotoxicity of Clematis chinensis Petroleum ether extract (Premna puberula root petroleum ether extract, P.puberula PEE) on human non-small cell lung cancer cell (A549) and normal cell human embryo lung fibroblast (MRC-5)
The experimental procedure used was complete medium, namely RPMI-1640 medium, containing 10% fetal bovine serum, 2mM glutamine, 100U/mL penicillin and 100. Mu.g/mL streptomycin. Selecting cells in logarithmic phase, performing pancreatin digestion, counting by a blood cell counting plate, and diluting to the required cell concentration by using RPMI-1640 complete culture medium; 80. Mu.L of the cells were plated in 96-well plates (5X 10 cells per well) 3 Individual cells), placed in CO 2 Culturing in a cell incubator for 24 hours; after the cells are completely stable or attached, 20 mu L of the petroleum ether extract of the falcate dolichos root is added into each hole, the RPMI-1640 culture medium is used for diluting the petroleum ether extract of the falcate dolichos root, the negative control group is an equal volume of culture medium, each group is provided with 5 parallel holes, the temperature is continuously 37 ℃ and the concentration of CO is 5 percent 2 Culturing in an incubator for 48 hours; after co-cultivation, 10. Mu.L MTT (5 mg/mL) was added to each well, followed by 5% CO at 37 ℃ 2 Incubating in an incubator for 4 hours; the 96-well plate was removed and the culture supernatant was aspirated. 150. Mu.L of DMSO was added and the mixture was shaken for 10min to allow complete dissolution of the MTT reaction product in DMSO. The OD value at 490nm wavelength was read with an ELISA reader, and the results were repeated three times to obtain an average value.
The inhibition rate was calculated as follows: cell strain inhibition ratio = [1- (sample group OD value-zeroed well OD value)/(control group OD value-zeroed well OD value) ] ×100%.
The concentration of the sample required to inhibit half cell proliferation (IC) was calculated using SPSS 25.0 analysis software 50 ) Using ICs 50 Samples were evaluated for cytotoxicity. The results are shown in Table 2, and the extract of the petroleum ether of the root of Buddha is useful for treating human non-small cell lung cancer cells (A549) (IC) 50 Cytotoxic activity was optimal for MRC-5 (IC) =38.01±3.36 μg/mL 50 = 76.85 ±3.18 μg/mL) is low. The inhibition of the extract of the petroleum ether of the falcate dolichos root on A549 and MRC-5 cells is compared, and the toxicity of the extract of the petroleum ether of the falcate dolichos root on normal cells is lower, so that the extract of the petroleum ether of the falcate dolichos root has better selectivity on cancer cells and has the characteristics of low toxicity and high anti-tumor activity.
Pharmacological example 2: effect of bromhidrosis root Petroleum Ether extract on A549 cell proliferation (plate cloning experiment)
Taking A549 logarithmic phase cells, and re-suspending the cells by using RPMI-1640 (containing 10% of fetal calf serum) culture solution after the cells are digested by 0.25% trypsin; counting by a blood cell counting plate, inoculating 200 cells/well into a 6-well plate, blowing dispersed cells, and placing in CO 2 Culturing in an incubator for 24 hours; the culture medium was discarded and 0, 5, 10, 15, 20, 25. Mu.g/mL of the medium was administered according to the blank group and the treatment group with the petroleum ether extract of Fagophus body odor, respectively. Three compound holes are arranged in each group, the conventional culture is continued, and after 2d, the fresh culture medium is replaced for continuous culture for 7d; observing under an inverted microscope, and checking the proliferation state and colony formation condition of the cells at any time until macroscopic cell colonies are formed; the medium was aspirated and washed twice with PBS, 800. Mu.L of fixative was added to each well, the fixative was discarded after 30min at room temperature, and 800. Mu.L of 0.1% crystal violet dye was added for staining. Placing for 15min in dark, discarding the dye liquor, washing the dye liquor with distilled water, and placing at room temperature to volatilize distilled water; the 6-well plate was placed under a white background cloth, counted after photographing, and its monoclonal ratio was calculated.
The influence of the bromhidrosis root petroleum ether extract on the formation of A549 cell colonies is examined through a plate cloning experiment, and the inhibition effect of the bromhidrosis root petroleum ether extract on the proliferation of the A549 cells is examined, and the result is shown in figure 2. The bromhidrosis falcate petroleum ether extract significantly reduced the number and size of a549 cell colonies compared to the blank group without bromhidrosis falcate petroleum ether extract treatment (fig. 2A). As shown in FIG. 2B, compared with the blank (clone formation rate: 43.50.+ -. 0.71%), the clone formation rates of A549 cells were reduced to 29.25.+ -. 1.06%, 20.00.+ -. 1.41%, 9.50.+ -. 0.71%, 4.75.+ -. 0.35%, 1.25.+ -. 0.35%, respectively, after the petroleum ether extract of Celastrus body, which was 5. Mu.g/mL, 10. Mu.g/mL, 15. Mu.g/mL, 20. Mu.g/mL and 25. Mu.g/mL was applied to the A549 cells for 7 days, indicating that the petroleum ether extract of Celastrus body significantly reduced the clone formation rate of A549 cells in a dose-dependent manner (p < 0.001). In conclusion, the bromhidrosis root petroleum ether extract obviously reduces the number and the size of colonies, obviously reduces the clone formation rate of A549 cells in a dose-dependent manner, and has obvious inhibition effect on the proliferation of the A549 cells.
Pharmacological example 3: effect of bromhidrosis root Petroleum Ether extract on A549 cell cycle
Collecting A549 logarithmic phase cells, counting by blood cell counting plate, and counting according to 4×10 5 Inoculating the cells/well into 6-well plate, and placing into CO 2 Culturing in an incubator for 24 hours; discarding the culture medium, and respectively feeding 0, 20, 40, 60, 80 and 100 mug/mL according to a blank group and a bromhidrosis root petroleum ether extract treatment group, wherein each group is provided with three compound holes, and continuing to perform conventional culture for 24 hours; cells were collected in EP tubes by trypsinization and the supernatant was discarded by centrifugation. Wash once with PBS and centrifuge to discard the supernatant. 1mL DNA Staining solution and 10 mu L Permeabilization solution are added and incubated for 30 minutes at room temperature in a dark place; red fluorescence at excitation wavelength 488nm was detected on a flow cytometer to analyze the phases of the cell cycle. The effect of the petroleum ether extract of falcate in the a549 cell cycle was examined by flow cytometry and the results are shown in figure 3. Compared with the blank group which is not treated by the bromhidrosis root petroleum ether extract, the bromhidrosis root petroleum ether extract obviously increases the proportion of S-phase cells, and simultaneously obviously downwards regulates the proportion of G1-phase cells and G2-phase cells, thereby leading to S-phase retardation. Compared with the blank (S phase: 37.53+ -0.83%), when 20 μg/mL, 40 μg/mL, 60 μg/mL, 80 μg/mL and 100 μg/mL of the Celastrus body petroleum ether extract were applied to the A549 cells for 24 hours, the proportion of the S phase cells of the A549 cells was increased to 39.88+ -0.74%, 46.08+ -0.14%, 50.62 + -0.96, 56.93+ -0.45 and 57.55 + -1.05%, respectively, indicating that the fraction of the S phase cells was significantly increased by the Celastrus body petroleum ether extract in a dose-dependent manner. Thus, the fraction of the bromhidrosis root petroleum ether extract significantly increases the S phase cells, resulting in S phase arrest of a549 cells.
Pharmacological example 4: effect of bromhidrosis root Petroleum Ether extract on A549 cell apoptosis
(1) Drug administration treatment: taking A549 logarithmic phase cells, and re-suspending the cells by using RPMI-1640 (containing 10% of fetal calf serum) culture solution after the cells are digested by 0.25% trypsin; counting by a blood cell counting plate, and then counting according to a ratio of 4×10 5 Inoculating the cells/well into 6-well plate, and placing into CO 2 Culturing in an incubator for 24 hours; the medium was discarded, 2mL of the medicated medium (0, 40, 60, 80 and 100. Mu.g/mL) was added, and the culture was continued for 48 hours.
(2) After the administration treatment, morphology changes of a549 cells were observed under an inverted microscope.
(3) After the treatment as in (1), AO/EB staining was performed. Mixing AO dye liquor and EB dye liquor according to the proportion of 1:1, and swirling to fully dissolve the AO dye liquor; the culture medium is discarded, the culture medium is gently washed twice by a precooled PBS buffer solution, 1mL of AO/EB dye solution is added into each hole, and the culture medium is incubated for 5min in a dark place; the dye solution was aspirated, observed under an inverted fluorescence microscope, photographed and analyzed.
(4) After the treatment of (1), hoechst 33258 staining was performed. Discarding the culture solution, adding 0.5mL of 4% paraformaldehyde solution fixing solution into each hole, fixing for 10min, discarding the fixing solution, washing twice with precooled PBS, and sucking the liquid; adding 0.5mL Hoechst 33258 staining solution into each hole, staining for 5min, discarding the staining solution, washing twice with precooled PBS, and sucking the solution; observed under an inverted fluorescence microscope, photographed and analyzed.
As shown in fig. 4A, after the a549 cells were treated with different concentrations of the bromhidrosis root petroleum ether extract for 48 hours, the shrunken cells increased significantly with the increase in the concentration of the bromhidrosis root petroleum ether extract; as shown in fig. 4B, after the treatment of the bromhidrosis root petroleum ether extracts with different concentrations, the proportion of green fluorescence gradually decreases, and meanwhile, the proportion of orange-red fluorescence gradually increases, which indicates that apoptotic cells gradually increase; as shown in fig. 4C, after the treatment of the bromhidrosis root petroleum ether extracts with different concentrations, the proportion of cells showing bright blue fluorescence of compact nuclei is gradually increased, and the characteristics of apoptosis are achieved. The morphological observations above all indicate that: the bromhidrosis root petroleum ether extract can effectively induce apoptosis of A549 cells.
The effect of the petroleum ether extract of the falcate dolichos root on inducing the apoptosis of A549 cells is further quantitatively detected by adopting an Annexin V-PE/7-AAD staining combined flow cytometry, and the result is shown in figure 5. Compared with the blank group (apoptosis rate: 7.04+ -0.95%), when 20. Mu.g/mL, 40. Mu.g/mL, 60. Mu.g/mL, 80. Mu.g/mL and 100. Mu.g/mL of the extract of Fagopyrum armpit root petroleum ether were applied to the A549 cells for 24 hours, the ratio of apoptosis of the A549 cells was increased to 17.68+ -0.72%, 58.66 + -0.87%, 66.52 + -0.76%, 86.60 + -0.08% and 95.73+ -0.54%, respectively, indicating that the extract of Fagopyrum armpit root petroleum ether significantly increased the ratio of apoptosis in a dose-dependent manner, while the extract of Fagopyrum armpit root petroleum ether significantly decreased the ratio of living cells. Thus, the bromhidrosis root petroleum ether extract significantly induced apoptosis of a549 cells in a dose-dependent manner.
Downregulation of mitochondrial membrane potential is a hallmark event of apoptosis. JC-1 is a fluorescent probe for detecting mitochondrial membrane potential (Δψm). When the mitochondrial membrane potential is high, JC-1 forms a polymer in the mitochondrial matrix, producing red fluorescence; when the mitochondrial membrane potential is low, JC-1 cannot form a polymer in the mitochondrial matrix, JC-1 is a monomer, and green fluorescence is generated. The Δψm value of mitochondria was measured by red-green fluorescence. The result of JC-1 staining is shown in FIG. 6. After the A549 cells are treated by the bromhidrosis root petroleum ether extracts with different concentrations for 48 hours, the proportion of red fluorescent cells is gradually reduced, and the proportion of green fluorescent cells is gradually increased. The results show that the bromhidrosis root petroleum ether extract can effectively lead to the down regulation of the mitochondrial membrane potential (delta psim) of A549 cells, thereby leading to apoptosis.
Key proteins in the mitochondrial mediated apoptosis pathway are detected by WB. Figures 7A and B depict levels of total Cyt c and mitochondrial Cyt c, which demonstrate that levels of mitochondrial Cyt c are significantly down-regulated in a dose-dependent manner following treatment with a petroleum ether extract of falcate root (40 μg/mL, 60 μg/mL and 80 μg/mL). In contrast, total Cyt c was significantly elevated, indicating that Cyt c was released from mitochondria. As shown in FIGS. 7C and D, the extract of the petroleum ether of the root of Buddha is up-regulated in Bax expression, while the ratio of Bax/Bcl-2 is inhibited from being significantly up-regulated in a dose-dependent manner. As shown in FIGS. 7C and E, the extract of Clematis chinensis root Petroleum ether significantly reduced the levels of pro-caspase 9 and pro-caspase 3, up-regulated the levels of clear-caspase 9, clear-caspase 3 and clear-PARP, indicating that the caspase cascade was activated, resulting in cleavage of PARP. Thus, the petroleum ether extract of falcate root can cause apoptosis of A549 cells through mitochondrial apoptosis pathway.
In conclusion, morphological observation, AO/EB staining, hoechst 33258 staining and apoptosis detection by a flow cytometer show that the A549 cell apoptosis is induced by the bromhidrosis root petroleum ether extract; JC-1 staining results show that the extract of the petroleum ether of the root of common bromhidrosis causes the down regulation of mitochondrial membrane potential (delta psim); the result of combining with the Western Blot related to mitochondrial apoptosis pathway shows that the mechanism of the bromhidrosis root petroleum ether extract for inducing A549 apoptosis is as follows: the extract of the petroleum ether of the root of Bungetree fern is up-regulated to the ratio of Bax/Bcl-2, thereby leading to the increase of the permeability of a mitochondrial membrane, the down regulation of the mitochondrial membrane potential (delta psi m), the release of Cyt C (Mitochondria Cyt C down regulation, total Cyt C up regulation) to cytoplasm by mitochondria, thereby cracking and activating Caspase-9 (Pro-Caspase 9 down regulation, clean-Caspase 9 up regulation), and then cutting and activating Caspase-3 (Pro-Caspase 3 down regulation, clean-Caspase 3 up regulation), and further the shearing and inactivating of PARP (clean-PARP up regulation), so as to lead to the apoptosis of A549.
Pharmacological example 5: effect of bromhidrosis root Petroleum Ether extract on A549 cell transfer
(1) Scratch experiment: the A549 cell suspension was collected and diluted to 2X 10 5 Individual cells/ml, then 4×10 were added to a six well plate 5 Individual cells. The cells were cultured overnight until the bottom of the six well plate was confluent to form a monolayer of confluent cells. A straight line was then drawn on the monolayer of cells per well with the tip of a 200. Mu.L pipette. The cells that have floated up due to streaking were then washed off with PBS, and then treated with various concentrations of Cepharanthus nikoense petroleum ether solution (formulated in serum-free medium) for 48 hours. The scratch areas were photographed at 0h and 48h with a Leka microscope. Cell mobility was calculated as follows:
(2) Invasion experiments: 750 μl of drug solution (15% FBS) of different concentrations was added to the lower chamber. 250 mu L A549 cell suspension (1X 10) 5 The cells/well) were inoculated into the upper chamber and 250. Mu.L of drug solutions of different concentrations (containing 5% FBS) were added. After 48h incubation, fixation with 10% paraformaldehyde for 2 min; incubating for 20 minutes with absolute methanol; 0.1% crystal violet staining for 15 min; after two PBS washes, images were taken with a Leka DMi8 microscope. The number of invading cells per field was quantified and statistically analyzed using ImageJ software.
Wound healing and invasion tests the effect of the petroleum ether extract of falcate in the presence of a549 cells on migration and invasiveness was studied. As shown in FIGS. 8A and B, the mobility of A549 cells treated with different concentrations (10, 20 and 30. Mu.g/mL) of the petroleum ether extract of Celastrus bromhidrosis was significantly reduced compared with the control group (73.92.+ -. 0.83%), 48.65.+ -. 1.29%, 34.30.+ -. 0.98% and 14.04.+ -. 1.51%, respectively. Therefore, the bromhidrosis root petroleum ether extract has obvious concentration dependence on the inhibition of the migration capacity of the A549 cells. The results of the invasion assay (fig. 8C and D) showed that the bromhidrosis root petroleum ether extract significantly reduced the invasion number of a549 cells compared to the control group, and that the dose-dependent, this showed that the bromhidrosis root petroleum ether extract inhibited the invasion capacity of a549 cells.
The influence of the bromhidrosis root petroleum ether extract on the transfer related protein is detected by WB. As shown in fig. 8E and F, the bromhidrosis root petroleum ether extract significantly reduced the expression level of MMP-2. In addition, it down-regulates the expression of N-cadherein in a concentration-dependent manner. The above results indicate that the bromhidrosis root petroleum ether extract inhibits the migration and invasive ability of a549 cells by down-regulating the levels of MMP-2 and N-cadherein.
By combining the pharmacological examples, the bromhidrosis root petroleum ether extract can block the cell cycle of A549 cells in the S phase so as to inhibit proliferation; the molecular mechanism of inducing apoptosis via a mitochondrial-mediated pathway is as follows: bax/Bcl-2 → Deltaψm → mitochondrial release Cyt C → Caspase-9 activation → Caspase-3 activation → PARP inactivation → induction of apoptosis; inhibition of migration and invasion of A549 cells is achieved by MMP-2 ∈ and N-cadhererin ∈.
Claims (7)
1. Application of bromhidrosis root petroleum ether extract in preparing medicine for treating non-small cell lung cancer is provided.
2. The petroleum ether extract of falcate dolichos root according to claim 1, prepared by the following method:
pulverizing fresh bromhidrosis root, mixing the pulverized raw materials with petroleum ether according to a feed-liquid ratio of 1:2-1:4, extracting the mixed materials in an extractor for 2-5 h, filtering, collecting petroleum ether extract, concentrating under reduced pressure, and removing solvent to obtain bromhidrosis root petroleum ether extract.
3. The use of claim 1, wherein the agent for treating non-small cell lung cancer is an agent that inhibits proliferation of non-small cell lung cancer cells.
4. The use of claim 1, wherein the agent for treating non-small cell lung cancer is an agent that induces apoptosis and inhibits cell cycle in non-small cell lung cancer cells.
5. The use of claim 1, wherein the agent that induces apoptosis and inhibits the cell cycle of non-small cell lung cancer is an agent that induces mitochondrial mediated apoptosis and causes S-phase arrest.
6. The use of claim 1, wherein the agent for treating non-small cell lung cancer is an agent that inhibits metastasis of non-small cell lung cancer cells.
7. The use of the petroleum ether extract of falcate dolichos root according to claim 1 for preparing a medicament for treating non-small cell lung cancer, wherein the petroleum ether extract of falcate dolichos root and a pharmaceutically acceptable carrier are prepared into a medicament in the form of tablets, capsules, fat emulsions, suppositories, dripping pills and ointments.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311218021.6A CN117244001A (en) | 2023-09-20 | 2023-09-20 | Application of bromhidrosis root petroleum ether extract in preparation of medicines for treating non-small cell lung cancer |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311218021.6A CN117244001A (en) | 2023-09-20 | 2023-09-20 | Application of bromhidrosis root petroleum ether extract in preparation of medicines for treating non-small cell lung cancer |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN117244001A true CN117244001A (en) | 2023-12-19 |
Family
ID=89136314
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202311218021.6A Pending CN117244001A (en) | 2023-09-20 | 2023-09-20 | Application of bromhidrosis root petroleum ether extract in preparation of medicines for treating non-small cell lung cancer |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN117244001A (en) |
-
2023
- 2023-09-20 CN CN202311218021.6A patent/CN117244001A/en active Pending
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Cochrane et al. | Anticancer effects of Annona glabra plant extracts in human leukemia cell lines | |
| Choene et al. | Validation of the antiproliferative effects of Euphorbia tirucalli extracts in breast cancer cell lines | |
| Ying et al. | Structural–activity relationship of ginsenosides from steamed ginseng in the treatment of erectile dysfunction | |
| CN112472729B (en) | Application of caulis sinomenii in preparing medicine for treating human glioma | |
| Liu et al. | Tanshinone IIA inhibits leukemia THP-1 cell growth by induction of apoptosis | |
| Zakaria et al. | Anti-uterine fibroid effect of standardized labisia pumila var. Alata extracts in vitro and in human uterine fibroid cancer xenograft model | |
| Wang et al. | Betulinic acid targets drug-resistant human gastric cancer cells by inducing autophagic cell death, suppresses cell migration and invasion, and modulates the ERK/MEK signaling pathway | |
| CN112274541B (en) | Application of semiliquidambar cathayensis aqueous extract in preparation of antitumor drugs | |
| Lin et al. | Anticancer effects of Taraxacum via cell cycle arrest, necrosis, apoptosis, and endoplasmic reticulum stress | |
| JP3294618B2 (en) | Harpagoside-rich extract from harpagofitsum procambens and method for producing the same | |
| WO2021142920A1 (en) | Traditional chinese medicine composition for treating lung cancer, and preparation and use thereof | |
| CN117244001A (en) | Application of bromhidrosis root petroleum ether extract in preparation of medicines for treating non-small cell lung cancer | |
| JI et al. | Antihepatocarcinoma effect of solanine and its mechanisms | |
| CN115590914B (en) | Euphorbia pekinensis extract and application thereof in preparation of breast cancer resisting medicines | |
| CN111743883A (en) | Benzophenone derivative composition, preparation method and application thereof | |
| Xia et al. | Chinese medicine formula “Shenqi San” extract inhibits proliferation of human lung adenocarcinoma A549 cells via inducing apoptosis | |
| WO2009100587A1 (en) | A drug composition for treatment and prevention of ischemic stroke and its preparation methods | |
| US8409632B2 (en) | Product containing extract from Zanthoxylum avicennae (Lam.) DC., and preparation process and use thereof | |
| Huang et al. | Improved Aitongxiao prescription (I-ATXP) induces apoptosis, cell cycle arrest and blocks exosomes release in hepatocellular carcinoma (HCC) cells | |
| CN111358804B (en) | Application of Cynoside H in preparation of medicine for preventing and treating breast cancer | |
| CN116139205A (en) | Pummelo peel extract and extraction method and application thereof | |
| Niu et al. | Phytochemical analysis and anticancer effect of Camellia oleifera bud ethanol extract in non-small cell lung cancer A549 cells | |
| Jia et al. | Anticancer effects of ovatodiolide on human prostate cancer cells involves cell cycle arrest, apoptosis and blocking of Ras/Raf/MEK/ERK signaling pathway | |
| CN113209241B (en) | Application of ginger volatile oil in preparation of medicine for treating non-small cell lung cancer | |
| CN109172549B (en) | Anti-tumor active composition and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |