WO2019218543A1 - Discovery and pharmaceutical use of equivalent component group of traditional chinese medicine rhizoma paridis yunnanensis, scutellaria indica, fructus xanthii sibirici, or bupleurum bicaule helm - Google Patents

Discovery and pharmaceutical use of equivalent component group of traditional chinese medicine rhizoma paridis yunnanensis, scutellaria indica, fructus xanthii sibirici, or bupleurum bicaule helm Download PDF

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WO2019218543A1
WO2019218543A1 PCT/CN2018/103855 CN2018103855W WO2019218543A1 WO 2019218543 A1 WO2019218543 A1 WO 2019218543A1 CN 2018103855 W CN2018103855 W CN 2018103855W WO 2019218543 A1 WO2019218543 A1 WO 2019218543A1
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composition
drug
saikosaponin
saponin
preparation
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PCT/CN2018/103855
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French (fr)
Chinese (zh)
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杨真慧
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Yang Zhenhui
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Priority claimed from CN201810457989.7A external-priority patent/CN108403682B/en
Priority claimed from CN201810458521.XA external-priority patent/CN108714147A/en
Priority claimed from CN201810458292.1A external-priority patent/CN108451966A/en
Priority claimed from CN201810457702.0A external-priority patent/CN108721312A/en
Priority claimed from CN201810457659.8A external-priority patent/CN108635364B/en
Priority claimed from CN201810457674.2A external-priority patent/CN108714150A/en
Priority claimed from CN201810456610.0A external-priority patent/CN108635363B/en
Priority claimed from CN201810458558.2A external-priority patent/CN108434168A/en
Application filed by Yang Zhenhui filed Critical Yang Zhenhui
Publication of WO2019218543A1 publication Critical patent/WO2019218543A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7048—Compounds having saccharide radicals and heterocyclic rings having oxygen as a ring hetero atom, e.g. leucoglucosan, hesperidin, erythromycin, nystatin, digitoxin or digoxin
    • 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)
    • A61K36/88—Liliopsida (monocotyledons)
    • A61K36/896—Liliaceae (Lily family), e.g. daylily, plantain lily, Hyacinth or narcissus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • the invention belongs to the field of medicine, relates to the discovery and pharmaceutical use of the equivalent component group of traditional Chinese medicine, and particularly relates to a composition for replacing the treatment of cervical cancer in the treatment of cervical cancer, and a medicine for treating ovarian cancer in the preparation of ovarian cancer.
  • Composition a composition for replacing Korean herbal medicine for preparing cervical cancer therapeutic drug, a composition for replacing Hanshin grass for preparing ovarian cancer therapeutic drug, and a compound for treating prostate cancer for replacing prostate cancer
  • Composition composition for treating prostate cancer therapeutic agent for replacing Xanthium sibiricum, composition for treating prostate cancer therapeutic agent for replacing Bupleurum chinense, and preparation for preparing brains for replacing Bupleurum chinense
  • a composition for the treatment of a stromal tumor A composition for the treatment of a stromal tumor.
  • Traditional Chinese medicine has the characteristics of multi-component, weak effect, and coordination and integration.
  • the overall effect of the drug is not a simple addition of single-component effects, but a multi-level, multi-link, multi-dimensional nonlinear effect between components.
  • the research group has established a set of basic research system for pharmacodynamic substances in line with the characteristics of traditional Chinese medicine: the analysis part in the whole, from partial regression to the whole, that is, on the basis of the overall composition of traditional Chinese medicine, following the proportion of the original ingredient content, evaluation
  • the contribution of “partial” to “whole” is to find “equivalent group of ingredients” that can basically represent the efficacy of the original side from many components.
  • the invention aims to provide the discovery and pharmaceutical use of the equivalent component group of traditional Chinese medicine, in particular to a composition for replacing the treatment of cervical cancer with the treatment of cervical cancer, and a combination for treating the preparation of ovarian cancer for the treatment of ovarian cancer.
  • Composition for treating cervical cancer therapeutic agent for replacing Hanshin grass, composition for replacing hanol cancer preparation ovarian cancer therapeutic agent, and composition for replacing prostate cancer preparation medicine for prostate cancer a composition for replacing a drug for treating prostate cancer of Xanthium sibiricum, a composition for replacing a drug for treating prostate cancer with Bupleurum chinense, and a method for preparing a glioma for replacing Bupleurum chinense A composition for treating a drug.
  • composition for the equivalent replacement of ⁇ in the preparation of a medicament for treating cervical cancer :
  • saponin VII From saponin VII, saponin-3-O- ⁇ -L-Rha(1 ⁇ 2)[ ⁇ -L-Rha(1 ⁇ 4)]- ⁇ -D-Glc, saponin H, heavy building Saponin II and fine diosgenin composition.
  • the above composition is used in the equivalent replacement of ⁇ to prepare a therapeutic drug for cervical cancer.
  • composition for the equivalent replacement of ⁇ in the preparation of a medicament for treating ovarian cancer :
  • saponin VII From saponin VII, saponin-3-O- ⁇ -L-Rha(1 ⁇ 2)[ ⁇ -L-Rha(1 ⁇ 4)]- ⁇ -D-Glc, saponin II, yam Saponin and heavy saponin I composition.
  • the above composition is used in the equivalent replacement of ⁇ to prepare a therapeutic drug for ovarian cancer.
  • a composition for equivalent replacement of Hanxincao for the preparation of a medicament for treating cervical cancer :
  • baicalin It is composed of wild baicalin, crude ragivin, baicalin, chrysin and apigenin.
  • the above composition is used in the equivalent replacement of Hanxincao for the preparation of a therapeutic drug for cervical cancer.
  • a composition for equivalent replacement of Hanxin grass for preparing ovarian cancer therapeutic drugs :
  • the above composition is used in the equivalent replacement of Hanxincao for the preparation of a medicament for treating ovarian cancer.
  • composition for the equivalent replacement of Xanthium for the preparation of a medicament for treating prostate cancer :
  • composition for the equivalent replacement of Xanthium sibiricum for preparing glioma therapeutic drugs :
  • a composition for the equivalent replacement of Bupleurum chinense to prepare a therapeutic drug for prostate cancer :
  • composition is used in the equivalent replacement of B. chinensis for the preparation of a medicament for treating prostate cancer.
  • a composition for the equivalent replacement of Bupleurum chinense to prepare glioma therapeutic drugs :
  • Embodiment 1 Equivalent component group of ⁇
  • the medicinal materials of ⁇ were purchased from the medicinal materials market in Zhangzhou.
  • the place of production was Lijiang, Yunnan. After smashing, it was dried overnight at 55 °C, and dried for storage.
  • RNAiso plus reagent SYBRGreen PCRMaster Mix and PrimeScript R kits were purchased from TaKaRa.
  • Saponin VII saponin-3-O- ⁇ -L-Rha(1 ⁇ 2)[ ⁇ -L-Rha(1 ⁇ 4)]- ⁇ -D-Glc(PGRR), saponin Determination of H, saponin II, saponin and saponin I in ⁇
  • the HPLC method includes the following chromatographic parameters:
  • Detection wavelength 210 nm.
  • Injection volume 20 ⁇ L.
  • Cervical adenocarcinoma HeLa cells and HO-8910 human ovarian cancer cells were subcultured in high glucose DMEM medium containing 10% fetal bovine serum at 37 ° C and 5% CO 2 . Logarithmic growth phase cells were used for experiments.
  • ⁇ The medicinal material of ⁇ was crushed, passed through a 40 mesh sieve, accurately weighed 10 g, placed in a round bottom flask, added 40 mL of 70% ethanol, refluxed for 2 h in a water bath, filtered, and the filtrate was taken and repeated 3 times. The filtrate was combined, evaporated to ethanol and dried to give a powder.
  • composition of the components of the composition drug is as follows, and each component is prepared by blending the standard product according to the content ratio of the sorghum building.
  • composition drug 1 5 Saponin VII, PGRR, saponin H, saponin II, saponin Composition drug 2 5 Saponins VII, PGRR, saponins II, saponins, and saponins I Composition drug 3 4 Saponin VII, saponin H, saponin II, and saponin Composition drug 4 4 Saponin VII, saponin II, saponin, and saponin I Composition drug 5 1 PGRR
  • the HeLa cell suspension at a concentration of 5.5 ⁇ 10 4 /mL was inoculated into a 96-well plate, and after 24 hours of culture, the drug was added to the drug, the composition drug 1, the composition drug 3, and the drug 5 to the final concentration.
  • the HO-8910 human ovarian cancer cell suspension with a concentration of 5.5 ⁇ 10 4 /mL was inoculated into a 96-well plate, and after 24 hours of culture, the drug was added to the drug, the composition drug 2, the composition drug 4, and the composition drug. 5 The final concentration was equivalent to 250 ⁇ g/mL of ⁇ , and a negative control group was set up.
  • Each group of 6 parallel wells was cultured for 48 hours in a 37 ° C, 5% CO 2 cell incubator, and the supernatant was centrifuged, PBS. Wash, collect cells, and set aside.
  • the upstream primer sequence of the p62 gene is 5'-TTTTCACTGTCAATTCACTGCA-3';
  • the p62 gene downstream primer sequence is 5'-GGAGGCGAGGATGGAGGAA-3';
  • the upstream primer sequence of the LSD1 gene is 5'-CAAGTGTCAATTTGTTCGGG-3';
  • the downstream primer sequence of the LSD1 gene is 5'-TTCTTTGGGCTGAGGTACTG-3';
  • the upstream primer sequence of the internal reference GAPDH gene is 5'-GCAAATTCCATGGCACCGTC-3';
  • the downstream primer sequence of the internal reference GAPDH gene is 5'-TCGCCCCACTTGATTTTGG-3'.
  • PCR reaction conditions 90 ° C for 30 s, 1 cycle; 95 ° C 5s, 60 ° C 30s, a total of 40 cycles.
  • the relative expression level of p62 mRNA and LSD1 mRNA is represented by a 2- ⁇ Ct value (Ct represents a cycle threshold). The experiment was repeated three times.
  • Saponin VII saponin-3-O- ⁇ -L-Rha(1 ⁇ 2)[ ⁇ -L-Rha(1 ⁇ 4)]- ⁇ -D-Glc(PGRR), saponin Determination of the content of H, saponin II, saponin and saponin I in ⁇
  • Saponin VII saponin-3-O- ⁇ -L-Rha(1 ⁇ 2)[ ⁇ -L-Rha(1 ⁇ 4)]- ⁇ -D-Glc(PGRR), saponin H,
  • saponin II, saponin, and saponin I in ⁇ are shown in the following table.
  • Compound name Content (mg/g) Compound name Content (mg/g) Saponin VII 26.25 Saponin II 30.46 PGRR 10.50 Slim yam 1.95 Saponin H 4.18 Saponin I 96.52
  • composition drug 1 composition drug 3
  • composition drug 5 The effects of ⁇ , composition drug 1, composition drug 3, and composition drug 5 on the expression level of p62 gene in HeLa cells are shown in the following table.
  • saponin H The composition consisting of saponin II and saponin saponin can basically replace the inhibitory effect of ⁇ on the expression of p62 gene, and the strength of the two is equivalent; the composition can be used for the equivalent replacement of 62 ⁇ to prepare p62 gene expression Inhibitors; those skilled in the art know that inhibition of p62 gene expression can effectively inhibit the proliferation of cervical cancer cells (RNA interference inhibits the expression of p62 gene on the proliferation of cervical cancer HeLa cells, Journal of Bengbu Medical College, Vol. 43, No. 4, January 2018 Period), therefore, the composition can be used for the equivalent replacement of ⁇ to prepare cervical cancer therapeutic drugs;
  • This phenomenon of 1+1 ⁇ 2 embodies the saponin-3-O- ⁇ -L-Rha(1 ⁇ 2)[ ⁇ -L-Rha(1) in the composition. ⁇ 4)]- ⁇ -D-Glc has an obvious synergistic effect on the expression of p62 gene in combination with the other four components.
  • saponin II From saponin VII, saponin-3-O- ⁇ -L-Rha(1 ⁇ 2)[ ⁇ -L-Rha(1 ⁇ 4)]- ⁇ -D-Glc, saponin II
  • the composition consisting of the saponin and the saponin I can basically replace the inhibitory effect of ⁇ on the expression of the LSD1 gene, and the strength of the two is equivalent; the composition can be used for the equivalent replacement of the LSD1 gene expression.
  • Inhibitors those skilled in the art know that inhibition of LSD1 gene expression can effectively inhibit the proliferation of ovarian cancer cells (silencing of LSD1 gene expression can inhibit the proliferation of ovarian cancer HO8910 cells and induce apoptosis, tumor, May 36, 36 Phase 5), therefore, the composition can be used for the equivalent replacement of ⁇ to prepare ovarian cancer therapeutic drugs;
  • This phenomenon of 1+1 ⁇ 2 embodies the saponin-3-O- ⁇ -L-Rha(1 ⁇ 2)[ ⁇ -L-Rha(1) in the composition. ⁇ 4)]- ⁇ -D-Glc has an obvious synergistic effect on the expression of the LSD1 gene in combination with the other four components.
  • Hanxin herbal medicines were purchased from the Chuzhou medicinal materials market. The origin was Sichuan, dried at 55 °C after smashing, and dried for storage.
  • RNAiso plus reagent SYBR Green PCR Master Mix and PrimeScript RT kit were purchased from TaKaRa.
  • the HPLC method includes the following chromatographic parameters:
  • Injection volume 20 ⁇ L.
  • Cervical adenocarcinoma HeLa cells and HO-8910 human ovarian cancer cells were subcultured in high glucose DMEM medium containing 10% fetal bovine serum at 37 ° C and 5% CO 2 . Logarithmic growth phase cells were used for experiments.
  • Hanxin grass medicine crush Hanxin herbal medicine, pass 40 mesh sieve, accurately weigh 10g, place it in a round bottom flask, add 40mL ethanol 40mL, reflux in water bath for 2h, filter, take the filtrate, repeat 3 times. The filtrate was combined, evaporated to ethanol and dried to give a powder.
  • composition drug The components of the composition drug are listed in the following table, and each component is prepared by blending a standard product according to the content ratio of Hanxin grass.
  • Composition drug 1 5 Wild baicalin, crude ragivin, baicalin, chrysin, apigenin Composition drug 2 5 Wild baicalin, crude ragivin, chrysin, apigenin, wogonin Composition drug 3 4 Wild baicalin, baicalin, chrysin, apigenin Composition drug 4 4 Wild baicalin, chrysin, apigenin, wogonin Composition drug 5 1 Corydalis
  • the HeLa cell suspension at a concentration of 5.5 ⁇ 10 4 /mL was inoculated into a 96-well plate, and after 24 hours of culture, the Hanshen drug, the composition drug 1, the composition drug 3, and the composition drug 5 were respectively added to make the final concentration equivalent.
  • the Hanxin herbal medicine was 1500 ⁇ g/mL, and the negative control group was set up.
  • Each group of 6 parallel wells was cultured in a 37 ° C, 5% CO 2 cell incubator for 72 h. The supernatant was centrifuged, washed with PBS, and the cells were collected for use.
  • the HO-8910 human ovarian cancer cell suspension with a concentration of 5.5 ⁇ 10 4 /mL was inoculated into a 96-well plate, and after 24 hours of culture, the Hanshin grass drug, the composition drug 2, the composition drug 4, and the composition drug 5 were respectively added.
  • the final concentration was equivalent to 1500 ⁇ g/mL of Hanxin herbal medicine, and a negative control group was set up.
  • Each group of 6 parallel wells was cultured in a 37 ° C, 5% CO 2 cell incubator for 72 h, then the supernatant was centrifuged, washed with PBS, Collect cells and set aside.
  • the upstream primer sequence of the p62 gene is 5'-TTTTCACTGTCAATTCACTGCA-3';
  • the p62 gene downstream primer sequence is 5'-GGAGGCGAGGATGGAGGAA-3';
  • the upstream primer sequence of the LSD1 gene is 5'-CAAGTGTCAATTTGTTCGGG-3';
  • the downstream primer sequence of the LSD1 gene is 5'-TTCTTTGGGCTGAGGTACTG-3';
  • the upstream primer sequence of the internal reference GAPDH gene is 5'-GCAAATTCCATGGCACCGTC-3';
  • the downstream primer sequence of the internal reference GAPDH gene is 5'-TCGCCCCACTTGATTTTGG-3'.
  • PCR reaction conditions 90 ° C for 30 s, 1 cycle; 95 ° C 5s, 60 ° C 30s, a total of 40 cycles.
  • the relative expression level of p62 mRNA and LSD1 mRNA is represented by a 2- ⁇ Ct value (Ct represents a cycle threshold). The experiment was repeated three times.
  • Compound name Content (mg/g) Compound name Content (mg/g) Wild baicalin 1.78 Chrysin 0.031 Corydalis 0.065 Apigenin 0.16 Hanknot 0.29 Hanaxanthin 0.13
  • composition consisting of wild baicalin, crude ragivin, baicalin, chrysin and apigenin can substantially replace the inhibitory effect of Hanxin grass on p62 gene expression, and the strength of the two is equivalent;
  • the composition can For the equivalent replacement of Hanxincao to prepare p62 gene expression inhibitor; those skilled in the art know that inhibition of p62 gene expression can effectively inhibit the proliferation of cervical cancer cells (RNA interference inhibits p62 gene expression on cervical cancer HeLa cell proliferation, ⁇ medicine Journal of the Academy of Sciences, Vol. 43, No. 1, January 2018), therefore, the composition can be used for the equivalent replacement of Hanxin grass for the preparation of cervical cancer therapeutic drugs;
  • a composition consisting of wild baicalin, crude ragivin, chrysin, apigenin and wogonin can substantially replace the inhibitory effect of Hanxin grass on the expression of LSD1 gene, and the strength of the two is equivalent;
  • For the equivalent replacement of Hanxin grass to prepare LSD1 gene expression inhibitor those skilled in the art know that inhibition of LSD1 gene expression can effectively inhibit the proliferation of ovarian cancer cells (silencing of LSD1 gene expression can inhibit the proliferation of ovarian cancer HO8910 cells and induce their withering Death, tumor, May 2016, Vol. 36, No. 5), therefore, the composition can be used for the equivalent replacement of Hanxin grass for the preparation of ovarian cancer therapeutic drugs;
  • the independent inhibition rate of LDD1 gene expression by burrowin is 10%, and the other 4 components are to LSD1.
  • the crude bursin and the other four components have an obvious synergistic effect of inhibiting the expression of the LSD1 gene.
  • Xanthium medicinal herbs are purchased from the Chuzhou medicinal materials market. The origin is Benxi, Liaoning. After smashing, it is dried overnight at 55 °C, and dried for storage.
  • RNAiso plus reagent SYBR Green PCR Master Mix and PrimeScript RT kit were purchased from TaKaRa.
  • the HPLC method includes the following chromatographic parameters:
  • Injection volume 20 ⁇ L.
  • the scorpion medicinal material was pulverized, passed through a 40 mesh sieve, and accurately weighed 10 g, placed in a round bottom flask, added with 40% ethanol 40 mL, refluxed in a water bath for 2 h, filtered, and the filtrate was taken and repeated three times. The filtrate was combined, evaporated to ethanol and dried to give a powder. The powder was dissolved in 250 mL of methanol and passed through a microporous membrane to obtain.
  • Human prostate cancer PC-3 cells were subcultured in F12 medium containing 10% fetal calf serum at 37 ° C under 5% CO 2 .
  • Glioblastoma U251 cells were subcultured in DMEM medium containing 10% fetal calf serum at 37 ° C under 5% CO 2 .
  • Logarithmic growth phase cells were used for experiments.
  • Xanthium drug The scorpion medicinal material is crushed, passed through a 40 mesh sieve, accurately weighed 10 g, placed in a round bottom flask, added 40 mL of 70% ethanol, refluxed for 2 h in a water bath, filtered, and the filtrate was taken and repeated 3 times. The filtrate was combined, evaporated to ethanol and dried to give a powder.
  • composition drug The components of the composition drug are listed in the following table, and each component is prepared by blending with a standard product according to the content ratio of Xanthium.
  • the human prostate cancer PC-3 cell suspension at a concentration of 5 ⁇ 10 4 /mL was inoculated into a 96-well plate, and after 24 hours of culture, the Xanthium drug, the composition drug 1, the composition drug 3, and the composition drug were respectively added. 5 The final concentration was equivalent to 2500 ⁇ g/mL of Xanthium sibiricum, and a negative control group was set up. Each group of 6 parallel wells was cultured in a 37 ° C, 5% CO 2 cell incubator for 72 h, then centrifuged to discard the supernatant, PBS. Wash, collect cells, and set aside.
  • the glioma U251 cell suspension at a concentration of 5 ⁇ 10 4 /mL was inoculated into a 96-well plate, and after 24 hours of culture, the Xanthium drug, the composition drug 2, the composition drug 4, and the composition drug 5 were respectively added.
  • the final concentration was equivalent to 2500 ⁇ g/mL of Xanthium sibiricum, and the negative control group was set up.
  • Each group of 6 parallel wells was cultured in a 37 ° C, 5% CO 2 cell incubator for 72 h, then the supernatant was centrifuged and washed with PBS. Collect cells and set aside.
  • qRT-PCR was used to detect the expression level of mTOR gene in AMPK gene and glioma U251 cells in human prostate cancer PC-3 cells.
  • the upstream primer sequence of the AMPK gene is 5'-TTGGTCAATGAAACCACTATCTG-3';
  • the downstream primer sequence of the AMPK gene is 5'-CGTTCGGCCATGATGTGTTAAGT-3';
  • the upstream primer sequence of the mTOR gene is 5'-GCGAACCTCAGGGCAAGAT-3';
  • the downstream primer sequence of the mTOR gene is 5'-TGACTCATCTCTCGGAGTTCCA-3';
  • the upstream primer sequence of the internal reference GAPDH gene is 5'-GCAAATTCCATGGCACCGTC-3';
  • the downstream primer sequence of the internal reference GAPDH gene is 5'-TCGCCCCACTTGATTTTGG-3'.
  • PCR reaction conditions 90 ° C for 30 s, 1 cycle; 95 ° C 5s, 60 ° C 30s, a total of 40 cycles.
  • the relative expression level of AMPK mRNA and mTOR mRNA is represented by a 2- ⁇ Ct value (Ct represents a cycle threshold). The experiment was repeated three times.
  • Compound name Content (mg/g) Compound name Content (mg/g) New chlorogenic acid 0.22 Thiazinium diketide 0.73 Chlorogenic acid 0.96 1,5-dicaffeoylquinic acid 1.34 Chlorogenic acid 0.67 4,5-dicaffeoylquinic acid 0.53
  • composition drug 1 composition drug 3
  • composition drug 5 The effects of Xanthium drug, composition drug 1, composition drug 3, and composition drug 5 on the expression level of AMPK gene in human prostate cancer PC-3 cells are shown in the following table.
  • a composition consisting of neochlorogenic acid, cryptochlorogenic acid, thiazinidine, 1,5-dicaffeoylquinic acid, 4,5-dicaffeoylquinic acid can be substantially substituted for Cang
  • the inhibitory effect of the ear on the expression of AMPK gene is equivalent; the composition can be used to prepare an inhibitor of AMPK gene expression by equivalent substitution of Xanthium; those skilled in the art know that inhibition of AMPK gene expression can effectively inhibit prostate cancer cells Proliferation (RNA interference silencing AMPK on the biological behavior of prostate cancer cells, Chinese Journal of Cancer Prevention and Treatment, Vol. 25, No. 2, January 2018), therefore, the composition can be used for the equivalent replacement of Xanthium in the preparation of prostate cancer drug;
  • composition drug 2 composition drug 4
  • composition drug 5 composition drug 5
  • a composition consisting of neochlorogenic acid, chlorogenic acid, thiazinidine, 1,5-dicaffeylquinic acid, 4,5-dicaffeoylquinic acid can be substantially substituted for Xanthium
  • the inhibitory effect of the sub-mTOR gene expression is equivalent to each other; the composition can be used for the equivalent replacement of Xanthium for the preparation of mTOR gene expression inhibitor; those skilled in the art know that inhibition of mTOR gene expression can effectively inhibit glioma Cell proliferation (miRNA-99b negatively regulates mTOR inhibition of glioma cell invasion, Chinese Journal of Pharmacology, April 2018, Vol. 34, No. 4), therefore, the composition can be used for equivalent replacement of Xanthium Preparation of glioma therapeutic drugs;
  • the medicinal material of B. chinensis was collected from Hailar, Inner Mongolia, dried at 55 ° C overnight, and dried for storage.
  • RNAiso plus reagent SYBR Green PCR Master Mix and PrimeScript RT kit were purchased from TaKaRa.
  • the HPLC method includes the following chromatographic parameters:
  • Detection wavelength 210 nm.
  • Injection volume 20 ⁇ L.
  • Human prostate cancer PC-3 cells were subcultured in F12 medium containing 10% fetal calf serum at 37 ° C under 5% CO 2 .
  • Glioblastoma U251 cells were subcultured in DMEM medium containing 10% fetal calf serum at 37 ° C under 5% CO 2 .
  • Logarithmic growth phase cells were used for experiments.
  • Cone Bupleurum medicine The medicinal material of B. chinensis was crushed, passed through a 40 mesh sieve, and accurately weighed 10 g, placed in a round bottom flask, added with 40 mL of ethanol 40 mL, refluxed for 2 h in a water bath, filtered, and the filtrate was taken and repeated 3 times. The filtrate was combined, evaporated to ethanol and dried to give a powder.
  • composition drug The components of the composition drug are listed in the following table, and each component is prepared by blending with a standard product according to the content ratio of Bupleurum chinense.
  • Composition drug 1 5 Bupleurum saponin F, saikosaponin I, saikosaponin C, saikosaponin A, saikosaponin D
  • Composition drug 2 5 Saikosaponin F, Chae saponin G, saikosaponin C, saikosaponin A, saikosaponin D
  • Composition drug 3 4 Saikosaponin F, Chae saponin I, saikosaponin A, saikosaponin D
  • Composition drug 5 1 Saikosaponin C
  • the human prostate cancer PC-3 cell suspension at a concentration of 5 ⁇ 10 4 /mL was inoculated into a 96-well plate, and after 24 hours of culture, the coniferous Bupleurum drug, the composition drug 1, the composition drug 3, and the composition were respectively added.
  • Drug 5 was made to have a final concentration equivalent to 400 ⁇ g/mL of B. chinensis, and a negative control group was set up.
  • Each group of 6 parallel wells was cultured in a 37 ° C, 5% CO 2 cell incubator for 48 h, and the supernatant was centrifuged. Wash with PBS, collect the cells, and set aside.
  • the glioma U251 cell suspension at a concentration of 5 ⁇ 10 4 /mL was inoculated into a 96-well plate, and after 24 hours of culture, the coniferous Bupleurum drug, the composition drug 2, the composition drug 4, and the composition drug were respectively added. 5 The final concentration was equivalent to 400 ⁇ g/mL of B. chinensis medicinal herbs, and a negative control group was set up. Each group of 6 parallel wells was cultured for 48 hours in a 37 ° C, 5% CO 2 cell incubator, and the supernatant was centrifuged. The cells were washed with PBS, and the cells were collected and used.
  • qRT-PCR was used to detect the expression level of mTOR gene in AMPK gene and glioma U251 cells in human prostate cancer PC-3 cells.
  • the upstream primer sequence of the AMPK gene is 5'-TTGGTCAATGAAACCACTATCTG-3';
  • the downstream primer sequence of the AMPK gene is 5'-CGTTCGGCCATGATGTGTTAAGT-3';
  • the upstream primer sequence of the mTOR gene is 5'-GCGAACCTCAGGGCAAGAT-3';
  • the downstream primer sequence of the mTOR gene is 5'-TGACTCATCTCTCGGAGTTCCA-3';
  • the upstream primer sequence of the internal reference GAPDH gene is 5'-GCAAATTCCATGGCACCGTC-3';
  • the downstream primer sequence of the internal reference GAPDH gene is 5'-TCGCCCCACTTGATTTTGG-3'.
  • PCR reaction conditions 90 ° C for 30 s, 1 cycle; 95 ° C 5s, 60 ° C 30s, a total of 40 cycles.
  • the relative expression level of AMPK mRNA and mTOR mRNA is represented by a 2- ⁇ Ct value (Ct represents a cycle threshold). The experiment was repeated three times.
  • Compound name Content (mg/g) Compound name Content (mg/g) Chae saponin F 8.83 Saikosaponin C 2.55 Chae saponin G 13.95 Saikosaponin A 3.76 Chaeminosaponin I 4.68 Saikosaponin D 17.50
  • the composition consisting of saikosaponin F, saikosaponin I, saikosaponin C, saikosaponin A, saikosaponin D can basically replace the inhibitory effect of Bupleurum chinense on AMPK gene expression.
  • the composition is equivalent in strength; the composition can be used to prepare an AMPK gene expression inhibitor equivalently to replace B. chinensis; those skilled in the art know that inhibition of AMPK gene expression can effectively inhibit proliferation of prostate cancer cells (RNA interference silencing AMPK pair)
  • the biological behavior of prostate cancer cells Chinese Journal of Cancer Prevention and Treatment, Vol. 25, No. 2, January 2018, therefore, the composition can be used for the equivalent replacement of Bupleurum chinense to prepare prostate cancer therapeutic drugs;
  • the composition consisting of saikosaponin F, saikosaponin G, saikosaponin C, saikosaponin A, saikosaponin D can basically replace the inhibitory effect of camphor bupleurum on mTOR gene expression.
  • the composition is equivalent in strength; the composition can be used to prepare an mTOR gene expression inhibitor equivalently to replace B. chinensis; those skilled in the art know that inhibition of mTOR gene expression can effectively inhibit the proliferation of glioma cells (miRNA-99b) Negative regulation of mTOR inhibits the invasive ability of glioma cells, Chinese Journal of Pharmacology, April 2018, Vol. 34, No. 4). Therefore, the composition can be used for the equivalent replacement of B. chinensis for the preparation of glioma. drug;

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Abstract

The present invention relates to a combination for replacing Rhizoma Paridis Yunnanensis to prepare a drug for treating cervical cancer, a combination for replacing Rhizoma Paridis Yunnanensis to prepare a drug for treating ovarian cancer, a combination for replacing Scutellaria indica to prepare a drug for treating cervical cancer, a combination for replacing Scutellaria indica to prepare a drug for treating ovarian cancer, a combination for replacing Fructus Xanthii Sibirici to prepare a drug for preparing prostate cancer, a combination for replacing Fructus Xanthii Sibirici to prepare a drug for preparing glioma, a combination for replacing Bupleurum bicaule Helm to prepare a drug for treating prostate cancer, and a combination for replacing Bupleurum bicaule Helm to prepare a drug for treating glioma.

Description

中药滇重楼、韩信草、苍耳子或锥叶柴胡等效成分群的发现与制药用途Discovery and pharmaceutical use of the equivalent component group of Chinese medicine 滇重楼, Hanxincao, Xanthium or Cone 技术领域Technical field
本发明属于医药领域，涉及中药等效成分群的发现与制药用途，具体涉及一种用于替代滇重楼制备宫颈癌治疗药物的组合物、一种用于替代滇重楼制备卵巢癌治疗药物的组合物、一种用于替代韩信草制备宫颈癌治疗药物的组合物、一种用于替代韩信草制备卵巢癌治疗药物的组合物、一种用于替代苍耳子制备前列腺癌治疗药物的组合物、一种用于替代苍耳子制备前列腺癌治疗药物的组合物、一种用于替代锥叶柴胡制备前列腺癌治疗药物的组合物、一种用于替代锥叶柴胡制备脑胶质瘤治疗药物的组合物。The invention belongs to the field of medicine, relates to the discovery and pharmaceutical use of the equivalent component group of traditional Chinese medicine, and particularly relates to a composition for replacing the treatment of cervical cancer in the treatment of cervical cancer, and a medicine for treating ovarian cancer in the preparation of ovarian cancer. Composition, a composition for replacing Korean herbal medicine for preparing cervical cancer therapeutic drug, a composition for replacing Hanshin grass for preparing ovarian cancer therapeutic drug, and a compound for treating prostate cancer for replacing prostate cancer Composition, composition for treating prostate cancer therapeutic agent for replacing Xanthium sibiricum, composition for treating prostate cancer therapeutic agent for replacing Bupleurum chinense, and preparation for preparing brains for replacing Bupleurum chinense A composition for the treatment of a stromal tumor.
背景技术Background technique
中药具有多成分、弱效应、协调整合作用特点，其整体药效的发挥不是单一成分药效的简单加合，而是存在着成分间多层次、多环节、多维度的非线性作用。基于此，该课题组建立了一套符合中药作用特点的药效物质基础研究体系：在整体中解析部分，由部分回归整体，即在中药复方整体的基础上，遵循原方成分含量比例，评估“部分”对“整体”的贡献，从众多成分中寻找能基本代表原方疗效的“等效成分群”。Traditional Chinese medicine has the characteristics of multi-component, weak effect, and coordination and integration. The overall effect of the drug is not a simple addition of single-component effects, but a multi-level, multi-link, multi-dimensional nonlinear effect between components. Based on this, the research group has established a set of basic research system for pharmacodynamic substances in line with the characteristics of traditional Chinese medicine: the analysis part in the whole, from partial regression to the whole, that is, on the basis of the overall composition of traditional Chinese medicine, following the proportion of the original ingredient content, evaluation The contribution of “partial” to “whole” is to find “equivalent group of ingredients” that can basically represent the efficacy of the original side from many components.
发明内容Summary of the invention
本发明旨在提供中药等效成分群的发现与制药用途，具体为一种用于替代滇重楼制备宫颈癌治疗药物的组合物、一种用于替代滇重楼制备卵巢癌治疗药物的组合物、一种用于替代韩信草制备宫颈癌治疗药物的组合物、一种用于替代韩信草制备卵巢癌治疗药物的组合物、一种用于替代苍耳子制备前列腺癌治疗药物的组合物、一种用于替代苍耳子制备前列腺癌治疗药物的组合物、一种用于替代锥叶柴胡制备前列腺癌治疗药物的组合物、一种用于替代锥叶柴胡制备脑胶质瘤治疗药物的组合物。The invention aims to provide the discovery and pharmaceutical use of the equivalent component group of traditional Chinese medicine, in particular to a composition for replacing the treatment of cervical cancer with the treatment of cervical cancer, and a combination for treating the preparation of ovarian cancer for the treatment of ovarian cancer. Composition for treating cervical cancer therapeutic agent for replacing Hanshin grass, composition for replacing hanol cancer preparation ovarian cancer therapeutic agent, and composition for replacing prostate cancer preparation medicine for prostate cancer a composition for replacing a drug for treating prostate cancer of Xanthium sibiricum, a composition for replacing a drug for treating prostate cancer with Bupleurum chinense, and a method for preparing a glioma for replacing Bupleurum chinense A composition for treating a drug.
本发明上述目的通过如下技术方案实现：The above object of the present invention is achieved by the following technical solutions:
一种用于等效替代滇重楼制备宫颈癌治疗药物的组合物：A composition for the equivalent replacement of 滇重楼 in the preparation of a medicament for treating cervical cancer:
由重楼皂苷VII、偏诺皂苷-3-O-α-L-Rha(1→2)[α-L-Rha(1→4)]-β-D-Glc、重楼皂苷H、重楼皂苷II和纤细薯蓣皂苷组成。From saponin VII, saponin-3-O-α-L-Rha(1→2)[α-L-Rha(1→4)]-β-D-Glc, saponin H, heavy building Saponin II and fine diosgenin composition.
上述组合物在等效替代滇重楼制备宫颈癌治疗药物方面的应用。The above composition is used in the equivalent replacement of 滇重楼 to prepare a therapeutic drug for cervical cancer.
一种用于等效替代滇重楼制备卵巢癌治疗药物的组合物：A composition for the equivalent replacement of 滇重楼 in the preparation of a medicament for treating ovarian cancer:
由重楼皂苷VII、偏诺皂苷-3-O-α-L-Rha(1→2)[α-L-Rha(1→4)]-β-D-Glc、重楼皂苷II、纤细薯蓣皂苷和重楼皂苷I组成。From saponin VII, saponin-3-O-α-L-Rha(1→2)[α-L-Rha(1→4)]-β-D-Glc, saponin II, yam Saponin and heavy saponin I composition.
上述组合物在等效替代滇重楼制备卵巢癌治疗药物方面的应用。The above composition is used in the equivalent replacement of 滇重楼 to prepare a therapeutic drug for ovarian cancer.
一种用于等效替代韩信草制备宫颈癌治疗药物的组合物：A composition for equivalent replacement of Hanxincao for the preparation of a medicament for treating cervical cancer:
由野黄芩苷、粗毛豚草素、汉黄芩苷、白杨素和芹菜素组成。It is composed of wild baicalin, crude ragivin, baicalin, chrysin and apigenin.
上述组合物在等效替代韩信草制备宫颈癌治疗药物方面的应用。The above composition is used in the equivalent replacement of Hanxincao for the preparation of a therapeutic drug for cervical cancer.
一种用于等效替代韩信草制备卵巢癌治疗药物的组合物：A composition for equivalent replacement of Hanxin grass for preparing ovarian cancer therapeutic drugs:
由野黄芩苷、粗毛豚草素、白杨素、芹菜素和汉黄芩素组成。It consists of wild baicalin, crude ragivin, chrysin, apigenin and wogonin.
上述组合物在等效替代韩信草制备卵巢癌治疗药物方面的应用。The above composition is used in the equivalent replacement of Hanxincao for the preparation of a medicament for treating ovarian cancer.
一种用于等效替代苍耳子制备前列腺癌治疗药物的组合物：A composition for the equivalent replacement of Xanthium for the preparation of a medicament for treating prostate cancer:
由新绿原酸、隐绿原酸、噻嗪双酮苷、1,5-二咖啡酰奎宁酸和4,5-二咖啡酰奎宁酸组成。It consists of neochlorogenic acid, cryptochlorogenic acid, thiazide diketide, 1,5-dicaffeoylquinic acid and 4,5-dicaffeoylquinic acid.
上述组合物在等效替代苍耳子制备前列腺癌治疗药物方面的应用。The use of the above composition for the equivalent replacement of Xanthium in the preparation of a medicament for treating prostate cancer.
一种用于等效替代苍耳子制备脑胶质瘤治疗药物的组合物：A composition for the equivalent replacement of Xanthium sibiricum for preparing glioma therapeutic drugs:
由新绿原酸、绿原酸、噻嗪双酮苷、1,5-二咖啡酰奎宁酸和4,5-二咖啡酰奎宁酸组成。It consists of neochlorogenic acid, chlorogenic acid, thiazide diketoside, 1,5-dicaffeoylquinic acid and 4,5-dicaffeoylquinic acid.
上述组合物在等效替代苍耳子制备脑胶质瘤治疗药物方面的应用。The use of the above composition for the equivalent replacement of Xanthium in the preparation of a medicament for the treatment of glioma.
一种用于等效替代锥叶柴胡制备前列腺癌治疗药物的组合物：A composition for the equivalent replacement of Bupleurum chinense to prepare a therapeutic drug for prostate cancer:
由柴胡次皂苷F、柴胡次皂苷I、柴胡皂苷C、柴胡皂苷A和柴胡皂苷D组成。It is composed of saikosaponin F, saikosaponin I, saikosaponin C, saikosaponin A and saikosaponin D.
上述组合物在等效替代锥叶柴胡制备前列腺癌治疗药物方面的应用。The above composition is used in the equivalent replacement of B. chinensis for the preparation of a medicament for treating prostate cancer.
一种用于等效替代锥叶柴胡制备脑胶质瘤治疗药物的组合物：A composition for the equivalent replacement of Bupleurum chinense to prepare glioma therapeutic drugs:
由柴胡次皂苷F、柴胡次皂苷G、柴胡皂苷C、柴胡皂苷A和柴胡皂苷D组成。It is composed of saikosaponin F, saikosaponin G, saikosaponin C, saikosaponin A and saikosaponin D.
上述组合物在等效替代锥叶柴胡制备脑胶质瘤治疗药物方面的应用。The use of the above composition in the preparation of a medicament for the treatment of glioma by an equivalent replacement of B. chinensis.
具体实施方式Detailed ways
下面结合实施例具体介绍本发明实质性内容，但并不以此限定本发明的保护范围。The substantial content of the present invention will be specifically described below with reference to the embodiments, but the scope of the present invention is not limited thereto.
实施例1：滇重楼等效成分群Embodiment 1: Equivalent component group of 滇重楼
一、实验材料First, the experimental materials
滇重楼药材购于亳州药材市场，产地为云南丽江，粉碎后55℃烘干过夜，干燥保存备用。The medicinal materials of 滇重楼 were purchased from the medicinal materials market in Zhangzhou. The place of production was Lijiang, Yunnan. After smashing, it was dried overnight at 55 °C, and dried for storage.
Agilent 1100液相色谱仪；Sartorius BS210S型电子天平，北京赛多利斯仪器系统有限公司；Sartorius CP225D型电子天平，北京赛多利斯仪器系统有限公司；乙腈(色谱纯)、甲醇(色谱纯)购自美国天地(TEDIA)；水为娃哈哈纯净水。Agilent 1100 liquid chromatograph; Sartorius BS210S electronic balance, Beijing Sartorius Instrument System Co., Ltd.; Sartorius CP225D electronic balance, Beijing Sartorius Instrument System Co., Ltd.; acetonitrile (chromatographically pure), methanol (chromatographically pure) purchased from American World (TEDIA); water is Wahaha pure water.
RNAiso plus试剂、SYBRGreen PCRMaster Mix和PrimeScript R试剂盒购自TaKaRa。RNAiso plus reagent, SYBRGreen PCRMaster Mix and PrimeScript R kits were purchased from TaKaRa.
二、实验方法Second, the experimental method
1、重楼皂苷VII、偏诺皂苷-3-O-α-L-Rha(1→2)[α-L-Rha(1→4)]-β-D-Glc(PGRR)、重楼皂苷H、重楼皂苷II、纤细薯蓣皂苷、重楼皂苷I在滇重楼中的含量测定1. Saponin VII, saponin-3-O-α-L-Rha(1→2)[α-L-Rha(1→4)]-β-D-Glc(PGRR), saponin Determination of H, saponin II, saponin and saponin I in 滇重楼
HPLC方法包括如下色谱参数：The HPLC method includes the following chromatographic parameters:
色谱仪：Agilent 1100液相色谱仪Chromatograph: Agilent 1100 Liquid Chromatograph
色谱柱：Agilent ZORBAX Extend-C18液相色谱柱(250mm×4.6mm，5μm)；Column: Agilent ZORBAX Extend-C18 liquid chromatography column (250 mm × 4.6 mm, 5 μm);
流动相：乙腈(A)-水(B)；Mobile phase: acetonitrile (A) - water (B);
洗脱程序：0～10min，20％～30％A；10～20min，30％～40％A；20～35min，40％～50％A；35～45min，50％～60％A；45～55min，60％～80％A；55～60min，80％～100％A；Elution procedure: 0 ~ 10min, 20% ~ 30% A; 10 ~ 20min, 30% ~ 40% A; 20 ~ 35min, 40% ~ 50% A; 35 ~ 45min, 50% ~ 60% A; 45 ~ 55 min, 60% to 80% A; 55 to 60 min, 80% to 100% A;
柱温：30℃。Column temperature: 30 ° C.
检测波长：210nm。Detection wavelength: 210 nm.
流动相流速：1.0mL/min。Mobile phase flow rate: 1.0 mL/min.
进样量：20μL。Injection volume: 20 μL.
供试品溶液的制备：将滇重楼药材粉碎，过40目筛，精密称取10g，置圆底烧瓶中，加入70％乙醇40mL，水浴回流2h，滤过，取滤液，重复3次。合并滤液，蒸去乙醇后干燥，得粉末。将粉末溶于250mL甲醇，过微孔滤膜，即得。Preparation of the test solution: The medicinal material of 滇重楼 was crushed, passed through a 40 mesh sieve, and accurately weighed 10 g, placed in a round bottom flask, added with 40% ethanol 40 mL, refluxed for 2 h in a water bath, filtered, and the filtrate was taken and repeated 3 times. The filtrate was combined, evaporated to ethanol and dried to give a powder. The powder was dissolved in 250 mL of methanol and passed through a microporous membrane to obtain.
精密量取供试品溶液20μL注入液相色谱仪，用标准品外标法测定重楼皂苷VII、偏诺皂苷-3-O-α-L-Rha(1→2)[α-L-Rha(1→4)]-β-D-Glc(PGRR)、重楼皂苷H、重楼皂苷II、纤细薯蓣皂苷、重楼皂苷I在滇重楼中的含量。Precisely take 20 μL of the test solution into the liquid chromatograph and determine the saponins VII and saponins-3-O-α-L-Rha(1→2)[α-L-Rha by standard external standard method. (1→4)]-β-D-Glc (PGRR), saponin H, saponin II, saponin, and saponin I in 滇重楼.
2、细胞培养2, cell culture
宫颈腺癌HeLa细胞、HO-8910人卵巢癌细胞分别在含10％胎牛血清的高糖DMEM培养基中，37℃、5％CO 2条件下传代培养。取对数生长期细胞用于实验。 Cervical adenocarcinoma HeLa cells and HO-8910 human ovarian cancer cells were subcultured in high glucose DMEM medium containing 10% fetal bovine serum at 37 ° C and 5% CO 2 . Logarithmic growth phase cells were used for experiments.
3、药物制备3, drug preparation
滇重楼药物：将滇重楼药材粉碎，过40目筛，精密称取10g，置圆底烧瓶中，加入70％乙醇40mL，水浴回流2h，滤过，取滤液，重复3次。合并滤液，蒸去乙醇后干燥，得粉末。滇重楼药: The medicinal material of 滇重楼 was crushed, passed through a 40 mesh sieve, accurately weighed 10 g, placed in a round bottom flask, added 40 mL of 70% ethanol, refluxed for 2 h in a water bath, filtered, and the filtrate was taken and repeated 3 times. The filtrate was combined, evaporated to ethanol and dried to give a powder.
组合物药物所含成分种类如下表，各成分按照在滇重楼的含量比例用标准品勾兑制得。The composition of the components of the composition drug is as follows, and each component is prepared by blending the standard product according to the content ratio of the sorghum building.
序号Serial number 数量Quantity 所含成分种类Type of ingredients
组合物药物1Composition drug 1 55 重楼皂苷VII、PGRR、重楼皂苷H、重楼皂苷II、纤细薯蓣皂苷Saponin VII, PGRR, saponin H, saponin II, saponin
组合物药物2Composition drug 2 55 重楼皂苷VII、PGRR、重楼皂苷II、纤细薯蓣皂苷、重楼皂苷ISaponins VII, PGRR, saponins II, saponins, and saponins I
组合物药物3Composition drug 3 44 重楼皂苷VII、重楼皂苷H、重楼皂苷II、纤细薯蓣皂苷Saponin VII, saponin H, saponin II, and saponin
组合物药物4Composition drug 4 44 重楼皂苷VII、重楼皂苷II、纤细薯蓣皂苷、重楼皂苷ISaponin VII, saponin II, saponin, and saponin I
组合物药物5Composition drug 5 11 PGRRPGRR
4、分组及给药4, grouping and drug delivery
将浓度为5.5×10 4/mL的HeLa细胞悬液接种于96孔板中，培养24h后，分别加入滇重楼药物、组合物药物1、组合物药物3、组合物药物5使其终浓度相当于滇重楼药材250μg/mL， 同时设阴性对照组，每组6个平行孔，37℃、5％CO 2细胞培养箱中培养48h后，离心弃上清液，PBS洗涤、收集细胞，备用。 The HeLa cell suspension at a concentration of 5.5×10 4 /mL was inoculated into a 96-well plate, and after 24 hours of culture, the drug was added to the drug, the composition drug 1, the composition drug 3, and the drug 5 to the final concentration. Equivalent to 250μg/mL of 滇重楼药, and a negative control group, 6 parallel holes in each group, cultured in 37°C, 5% CO 2 cell incubator for 48h, centrifuged the supernatant, washed with PBS, and collected the cells. spare.
将浓度为5.5×10 4/mL的HO-8910人卵巢癌细胞悬液接种于96孔板中，培养24h后，分别加入滇重楼药物、组合物药物2、组合物药物4、组合物药物5使其终浓度相当于滇重楼药材250μg/mL，同时设阴性对照组，每组6个平行孔，37℃、5％CO 2细胞培养箱中培养48h后，离心弃上清液，PBS洗涤、收集细胞，备用。 The HO-8910 human ovarian cancer cell suspension with a concentration of 5.5×10 4 /mL was inoculated into a 96-well plate, and after 24 hours of culture, the drug was added to the drug, the composition drug 2, the composition drug 4, and the composition drug. 5 The final concentration was equivalent to 250 μg/mL of 滇重楼药, and a negative control group was set up. Each group of 6 parallel wells was cultured for 48 hours in a 37 ° C, 5% CO 2 cell incubator, and the supernatant was centrifuged, PBS. Wash, collect cells, and set aside.
5、qRT-PCR测定HeLa细胞p62基因和HO-8910人卵巢癌细胞LSD1基因的表达水平5, qRT-PCR determination of HeLa cell p62 gene and HO-8910 human ovarian cancer cell LSD1 gene expression level
收集上述细胞，用PBS洗涤细胞3次，按照RNAiso Plus试剂盒操作手册提取细胞总RNA，并用紫外分光光度法行定量分析。采用PrimeScript RT试剂盒将RNA反转录为cDNA，以cDNA为模板，采用SYBR-Green PCR Master Mix试剂盒进一步行PCR扩增。The above cells were collected, and the cells were washed 3 times with PBS, and total RNA was extracted according to the RNAiso Plus kit operating manual, and quantitative analysis was carried out by ultraviolet spectrophotometry. The RNA was reverse transcribed into cDNA using the PrimeScript RT kit, and cDNA amplification was carried out using the SYBR-Green PCR Master Mix kit using cDNA as a template.
p62基因上游引物序列为5’-TTTTCACTGTCAATTCACTGCA-3’；The upstream primer sequence of the p62 gene is 5'-TTTTCACTGTCAATTCACTGCA-3';
p62基因下游引物序列为5’-GGAGGCGAGGATGGAGGAA-3’；The p62 gene downstream primer sequence is 5'-GGAGGCGAGGATGGAGGAA-3';
LSD1基因上游引物序列为5’-CAAGTGTCAATTTGTTCGGG-3’；The upstream primer sequence of the LSD1 gene is 5'-CAAGTGTCAATTTGTTCGGG-3';
LSD1基因下游引物序列为5’-TTCTTTGGGCTGAGGTACTG-3’；The downstream primer sequence of the LSD1 gene is 5'-TTCTTTGGGCTGAGGTACTG-3';
内参GAPDH基因上游引物序列为5’-GCAAATTCCATGGCACCGTC-3’；The upstream primer sequence of the internal reference GAPDH gene is 5'-GCAAATTCCATGGCACCGTC-3';
内参GAPDH基因下游引物序列为5’-TCGCCCCACTTGATTTTGG-3’。The downstream primer sequence of the internal reference GAPDH gene is 5'-TCGCCCCACTTGATTTTGG-3'.
PCR反应条件：90℃30s，1个循环；95℃5s、60℃30s，共40个循环。以2 -ΔΔCt值(Ct代表循环阈值)表示p62mRNA、LSD1mRNA的相对表达量。实验重复3次。 PCR reaction conditions: 90 ° C for 30 s, 1 cycle; 95 ° C 5s, 60 ° C 30s, a total of 40 cycles. The relative expression level of p62 mRNA and LSD1 mRNA is represented by a 2- ΔΔCt value (Ct represents a cycle threshold). The experiment was repeated three times.
6、统计学方法6, statistical methods
采用Graph PadPrism 5软件进行数据的统计和分析。计量资料以x±s表示，多组间比较采用单因素方差分析，两两比较采用Tukey检验。P＜0.05为差异有统计学意义。Data statistics and analysis were performed using Graph PadPrism 5 software. The measurement data were expressed as x±s, and the comparison between groups was performed by one-way analysis of variance, and the comparison between the two groups was performed by Tukey test. P < 0.05 was considered statistically significant.
三、实验结果Third, the experimental results
1、重楼皂苷VII、偏诺皂苷-3-O-α-L-Rha(1→2)[α-L-Rha(1→4)]-β-D-Glc(PGRR)、重楼皂苷H、重楼皂苷II、纤细薯蓣皂苷、重楼皂苷I在滇重楼中的含量测定结果1. Saponin VII, saponin-3-O-α-L-Rha(1→2)[α-L-Rha(1→4)]-β-D-Glc(PGRR), saponin Determination of the content of H, saponin II, saponin and saponin I in 滇重楼
重楼皂苷VII、偏诺皂苷-3-O-α-L-Rha(1→2)[α-L-Rha(1→4)]-β-D-Glc(PGRR)、重楼皂苷H、重楼皂苷II、纤细薯蓣皂苷、重楼皂苷I在滇重楼中的含量如下表所示。Saponin VII, saponin-3-O-α-L-Rha(1→2)[α-L-Rha(1→4)]-β-D-Glc(PGRR), saponin H, The contents of saponin II, saponin, and saponin I in 滇重楼 are shown in the following table.
化合物名称Compound name 含量(mg/g)Content (mg/g) 化合物名称Compound name 含量(mg/g)Content (mg/g)
重楼皂苷VIISaponin VII 26.2526.25 重楼皂苷IISaponin II 30.4630.46
PGRRPGRR 10.5010.50 纤细薯蓣皂苷Slim yam 1.951.95
重楼皂苷HSaponin H 4.184.18 重楼皂苷ISaponin I 96.5296.52
2、滇重楼药物及组合物对HeLa细胞p62基因的表达水平的影响2. Effect of drugs and compositions of Suizhonglou on the expression level of p62 gene in HeLa cells
滇重楼药物、组合物药物1、组合物药物3、组合物药物5对HeLa细胞p62基因的表达水平的影响如下表所示。The effects of 滇重楼药, composition drug 1, composition drug 3, and composition drug 5 on the expression level of p62 gene in HeLa cells are shown in the following table.
组别Group p62mRNA/GAPDHmRNAp62mRNA/GAPDH mRNA 抑制率Inhibition rate
阴性对照组Negative control group 1.001.00 //
滇重楼药物(相当于滇重楼药材250μg/mL)滇重楼药(equivalent to 250μg/mL of 滇重楼药) 0.450.45 55％55%
组合物药物1(相当于滇重楼药材250μg/mL)Composition drug 1 (equivalent to 250μg/mL of 滇重楼药) 0.470.47 53％53%
组合物药物3(相当于滇重楼药材250μg/mL)Composition drug 3 (equivalent to 250μg/mL of 滇重楼药) 0.810.81 19％19%
组合物药物5(相当于滇重楼药材250μg/mL)Composition drug 5 (equivalent to 250μg/mL of 滇重楼药) 0.890.89 11％11%
上述实验结果说明：The above experimental results show that:
(1)由重楼皂苷VII、偏诺皂苷-3-O-α-L-Rha(1→2)[α-L-Rha(1→4)]-β-D-Glc、重楼皂苷H、重楼皂苷II和纤细薯蓣皂苷组成的组合物基本可以等效替代滇重楼对p62基因表达的抑制效应，二者强度相当；该组合物可以用于等效替代滇重楼制备p62基因表达抑制剂；本领域技术人员知道，抑制p62基因表达可以有效抑制宫颈癌细胞的增殖(RNA干扰抑制p62基因表达对宫颈癌HeLa细胞增殖的影响，蚌埠医学院学报2018年1月第43卷第1期)，因此，该组合物可以用于等效替代滇重楼制备宫颈癌治疗药物；(1) From saponin VII, saponin-3-O-α-L-Rha(1→2)[α-L-Rha(1→4)]-β-D-Glc, saponin H The composition consisting of saponin II and saponin saponin can basically replace the inhibitory effect of 滇重楼 on the expression of p62 gene, and the strength of the two is equivalent; the composition can be used for the equivalent replacement of 62重楼 to prepare p62 gene expression Inhibitors; those skilled in the art know that inhibition of p62 gene expression can effectively inhibit the proliferation of cervical cancer cells (RNA interference inhibits the expression of p62 gene on the proliferation of cervical cancer HeLa cells, Journal of Bengbu Medical College, Vol. 43, No. 4, January 2018 Period), therefore, the composition can be used for the equivalent replacement of 滇重楼 to prepare cervical cancer therapeutic drugs;
(2)由重楼皂苷VII、偏诺皂苷-3-O-α-L-Rha(1→2)[α-L-Rha(1→4)]-β-D-Glc、重楼皂苷H、重楼皂苷II和纤细薯蓣皂苷组成的组合物中，偏诺皂苷-3-O-α-L-Rha(1→2)[α-L-Rha(1→4)]-β-D-Glc对p62基因表达的独立抑制率是11％，其他4种成分对p62基因表达的组合抑制率是19％，二者之和(11％+19％＝30％)显著小于5种成分对p62基因表达的组合抑制率53％，这种1+1＜2的现象体现了该组合物中偏诺皂苷-3-O-α-L-Rha(1→2)[α-L-Rha(1→4)]-β-D-Glc与其他4种成分存在明显的协同抑制p62基因表达的效应。(2) From saponin VII, saponin-3-O-α-L-Rha(1→2)[α-L-Rha(1→4)]-β-D-Glc, saponin H In the composition consisting of saponin II and saponin, the saponin-3-O-α-L-Rha(1→2)[α-L-Rha(1→4)]-β-D- The independent inhibition rate of plc gene expression by Glc was 11%, and the combined inhibition rate of p62 gene expression by the other four components was 19%, and the sum of the two (11%+19%=30%) was significantly less than 5 components to p62. The combined inhibition rate of gene expression is 53%. This phenomenon of 1+1<2 embodies the saponin-3-O-α-L-Rha(1→2)[α-L-Rha(1) in the composition. →4)]-β-D-Glc has an obvious synergistic effect on the expression of p62 gene in combination with the other four components.
3、滇重楼药物及组合物对HO-8910人卵巢癌细胞LSD1基因的表达水平的影响3. Effect of drugs and compositions of 滇重楼 on the expression level of LSD1 gene in HO-8910 human ovarian cancer cells
滇重楼药物、组合物药物2、组合物药物4、组合物药物5对HO-8910人卵巢癌细胞LSD1基因的表达水平的影响如下表所示。The effects of 滇重楼药, Composition Drug 2, Composition Drug 4, and Composition Drug 5 on the expression level of HO-8910 human ovarian cancer cell LSD1 gene are shown in the following table.
组别Group LSD1mRNA/GAPDHmRNALSD1 mRNA/GAPDH mRNA 抑制率Inhibition rate
阴性对照组Negative control group 1.001.00 //
滇重楼药物(相当于滇重楼药材250μg/mL)滇重楼药(equivalent to 250μg/mL of 滇重楼药) 0.530.53 47％47%
组合物药物2(相当于滇重楼药材250μg/mL)Composition drug 2 (equivalent to 250μg/mL of 滇重楼药) 0.520.52 48％48%
组合物药物4(相当于滇重楼药材250μg/mL)Composition drug 4 (equivalent to 250μg/mL of 滇重楼药) 0.780.78 22％twenty two%
组合物药物5(相当于滇重楼药材250μg/mL)Composition drug 5 (equivalent to 250μg/mL of 滇重楼药) 0.880.88 12％12%
上述实验结果说明：The above experimental results show that:
(1)由重楼皂苷VII、偏诺皂苷-3-O-α-L-Rha(1→2)[α-L-Rha(1→4)]-β-D-Glc、重楼皂苷II、纤细薯蓣皂苷和重楼皂苷I组成的组合物基本可以等效替代滇重楼对LSD1基因表达的抑制效应，二者强度相当；该组合物可以用于等效替代滇重楼制备LSD1基因表达抑制剂；本领域技术人员知道，抑制LSD1基因表达可以有效抑制卵巢癌细胞的增殖(沉默LSD1基因的表达可抑制卵巢癌HO8910细胞的增殖并诱导其凋亡，肿瘤2016年5月第36卷第5期)，因此，该组合物可以用于等效替代滇重楼制备卵巢癌治疗药物；(1) From saponin VII, saponin-3-O-α-L-Rha(1→2)[α-L-Rha(1→4)]-β-D-Glc, saponin II The composition consisting of the saponin and the saponin I can basically replace the inhibitory effect of 滇重楼 on the expression of the LSD1 gene, and the strength of the two is equivalent; the composition can be used for the equivalent replacement of the LSD1 gene expression. Inhibitors; those skilled in the art know that inhibition of LSD1 gene expression can effectively inhibit the proliferation of ovarian cancer cells (silencing of LSD1 gene expression can inhibit the proliferation of ovarian cancer HO8910 cells and induce apoptosis, tumor, May 36, 36 Phase 5), therefore, the composition can be used for the equivalent replacement of 滇重楼 to prepare ovarian cancer therapeutic drugs;
(2)由重楼皂苷VII、偏诺皂苷-3-O-α-L-Rha(1→2)[α-L-Rha(1→4)]-β-D-Glc、重楼皂苷II、纤细薯蓣皂苷和重楼皂苷I组成的组合物中，偏诺皂苷-3-O-α-L-Rha(1→2)[α-L-Rha(1→4)]-β-D-Glc对LSD1基因表达的独立抑制率是12％，其他4种成分对LSD1基因表达的组合抑制率是22％，二者之和(12％+22％＝34％)显著小于5种成分对LSD1基因表达的组合抑制率48％，这种1+1＜2的现象体现了该组合物中偏诺皂苷-3-O-α-L-Rha(1→2)[α-L-Rha(1→4)]-β-D-Glc与其他4种成分存在明显的协同抑制LSD1基因表达的效应。(2) From saponin VII, saponin-3-O-α-L-Rha(1→2)[α-L-Rha(1→4)]-β-D-Glc, saponin II In a composition consisting of saponin and saponin I, saponin-3-O-α-L-Rha(1→2)[α-L-Rha(1→4)]-β-D- The independent inhibition rate of Glc on LSD1 gene expression was 12%, and the combined inhibition rate of LSD1 gene expression by the other four components was 22%, and the sum of the two (12%+22%=34%) was significantly less than 5 components to LSD1. The combined inhibition rate of gene expression is 48%. This phenomenon of 1+1<2 embodies the saponin-3-O-α-L-Rha(1→2)[α-L-Rha(1) in the composition. →4)]-β-D-Glc has an obvious synergistic effect on the expression of the LSD1 gene in combination with the other four components.
实施例2：韩信草等效成分群Example 2: Han Xincao equivalent component group
一、实验材料First, the experimental materials
韩信草药材购于亳州药材市场，产地为四川，粉碎后55℃烘干过夜，干燥保存备用。Hanxin herbal medicines were purchased from the Chuzhou medicinal materials market. The origin was Sichuan, dried at 55 °C after smashing, and dried for storage.
Agilent 1100液相色谱仪；Sartorius BS210S型电子天平，北京赛多利斯仪器系统有限公司；Sartorius CP225D型电子天平，北京赛多利斯仪器系统有限公司；乙腈(色谱纯)、甲醇(色谱纯)购自美国天地(TEDIA)；水为娃哈哈纯净水。Agilent 1100 liquid chromatograph; Sartorius BS210S electronic balance, Beijing Sartorius Instrument System Co., Ltd.; Sartorius CP225D electronic balance, Beijing Sartorius Instrument System Co., Ltd.; acetonitrile (chromatographically pure), methanol (chromatographically pure) purchased from American World (TEDIA); water is Wahaha pure water.
RNAiso plus试剂、SYBRGreen PCRMaster Mix和PrimeScript RT试剂盒购自TaKaRa。RNAiso plus reagent, SYBR Green PCR Master Mix and PrimeScript RT kit were purchased from TaKaRa.
二、实验方法Second, the experimental method
1、野黄芩苷、粗毛豚草素、汉黄芩苷、白杨素、芹菜素、汉黄芩素在韩信草中含量测定1. Determination of the content of wild baicalin, crude ragivin, baicalin, chrysin, apigenin and wogonin in Hanxincao
HPLC方法包括如下色谱参数：The HPLC method includes the following chromatographic parameters:
色谱仪：Agilent 1100液相色谱仪Chromatograph: Agilent 1100 Liquid Chromatograph
色谱柱：Kromasil 100-5-C18液相色谱柱(250mm×4.6mm，5μm)；Column: Kromasil 100-5-C18 liquid chromatography column (250 mm × 4.6 mm, 5 μm);
流动相：乙腈(A)-0.3％冰乙酸溶液(B)；Mobile phase: acetonitrile (A) - 0.3% glacial acetic acid solution (B);
洗脱程序：0～20min，10％～20％A；20～30min，20％～30％A；30～50min，30％～50％A；50～60min，50％A；Elution procedure: 0 ~ 20min, 10% ~ 20% A; 20 ~ 30min, 20% ~ 30% A; 30 ~ 50min, 30% ~ 50% A; 50 ~ 60min, 50% A;
柱温：35℃。Column temperature: 35 ° C.
检测波长：280nm。Detection wavelength: 280 nm.
流动相流速：1.0mL/min。Mobile phase flow rate: 1.0 mL/min.
进样量：20μL。Injection volume: 20 μL.
供试品溶液的制备：将韩信草药材粉碎，过40目筛，精密称取10g，置圆底烧瓶中，加入70％乙醇40mL，水浴回流2h，滤过，取滤液，重复3次。合并滤液，蒸去乙醇后干燥，得粉末。将粉末溶于250mL甲醇，过微孔滤膜，即得。Preparation of the test solution: The Hanxin herbal medicine was pulverized, passed through a 40-mesh sieve, and accurately weighed 10 g, placed in a round bottom flask, added with 40% ethanol 40 mL, refluxed in a water bath for 2 h, filtered, and the filtrate was taken, and repeated three times. The filtrate was combined, evaporated to ethanol and dried to give a powder. The powder was dissolved in 250 mL of methanol and passed through a microporous membrane to obtain.
精密量取供试品溶液20μL注入液相色谱仪，用标准品外标法测定野黄芩苷、粗毛豚草素、汉黄芩苷、白杨素、芹菜素、汉黄芩素在韩信草中的含量。Precisely take 20 μL of the test solution into the liquid chromatograph, and determine the content of wild baicalin, crude ragivin, baicalin, chrysin, apigenin, and wogonin in Hanxin grass by standard external standard method.
2、细胞培养2, cell culture
宫颈腺癌HeLa细胞、HO-8910人卵巢癌细胞分别在含10％胎牛血清的高糖DMEM培养基中，37℃、5％CO 2条件下传代培养。取对数生长期细胞用于实验。 Cervical adenocarcinoma HeLa cells and HO-8910 human ovarian cancer cells were subcultured in high glucose DMEM medium containing 10% fetal bovine serum at 37 ° C and 5% CO 2 . Logarithmic growth phase cells were used for experiments.
3、药物制备3, drug preparation
韩信草药物：将韩信草药材粉碎，过40目筛，精密称取10g，置圆底烧瓶中，加入70％乙醇40mL，水浴回流2h，滤过，取滤液，重复3次。合并滤液，蒸去乙醇后干燥，得粉末。Hanxin grass medicine: crush Hanxin herbal medicine, pass 40 mesh sieve, accurately weigh 10g, place it in a round bottom flask, add 40mL ethanol 40mL, reflux in water bath for 2h, filter, take the filtrate, repeat 3 times. The filtrate was combined, evaporated to ethanol and dried to give a powder.
组合物药物所含成分种类如下表，各成分按照在韩信草的含量比例用标准品勾兑制得。The components of the composition drug are listed in the following table, and each component is prepared by blending a standard product according to the content ratio of Hanxin grass.
序号Serial number 数量Quantity 所含成分种类Type of ingredients
组合物药物1Composition drug 1 55 野黄芩苷、粗毛豚草素、汉黄芩苷、白杨素、芹菜素Wild baicalin, crude ragivin, baicalin, chrysin, apigenin
组合物药物2Composition drug 2 55 野黄芩苷、粗毛豚草素、白杨素、芹菜素、汉黄芩素Wild baicalin, crude ragivin, chrysin, apigenin, wogonin
组合物药物3Composition drug 3 44 野黄芩苷、汉黄芩苷、白杨素、芹菜素Wild baicalin, baicalin, chrysin, apigenin
组合物药物4Composition drug 4 44 野黄芩苷、白杨素、芹菜素、汉黄芩素Wild baicalin, chrysin, apigenin, wogonin
组合物药物5Composition drug 5 11 粗毛豚草素Corydalis
4、分组及给药4, grouping and drug delivery
将浓度为5.5×10 4/mL的HeLa细胞悬液接种于96孔板中，培养24h后，分别加入韩信草药物、组合物药物1、组合物药物3、组合物药物5使其终浓度相当于韩信草药材1500μg/mL，同时设阴性对照组，每组6个平行孔，37℃、5％CO 2细胞培养箱中培养72h后，离心弃上清液，PBS洗涤、收集细胞，备用。 The HeLa cell suspension at a concentration of 5.5×10 4 /mL was inoculated into a 96-well plate, and after 24 hours of culture, the Hanshen drug, the composition drug 1, the composition drug 3, and the composition drug 5 were respectively added to make the final concentration equivalent. The Hanxin herbal medicine was 1500 μg/mL, and the negative control group was set up. Each group of 6 parallel wells was cultured in a 37 ° C, 5% CO 2 cell incubator for 72 h. The supernatant was centrifuged, washed with PBS, and the cells were collected for use.
将浓度为5.5×10 4/mL的HO-8910人卵巢癌细胞悬液接种于96孔板中，培养24h后，分别加入韩信草药物、组合物药物2、组合物药物4、组合物药物5使其终浓度相当于韩信草药材1500μg/mL，同时设阴性对照组，每组6个平行孔，37℃、5％CO 2细胞培养箱中培养72h后，离心弃上清液，PBS洗涤、收集细胞，备用。 The HO-8910 human ovarian cancer cell suspension with a concentration of 5.5×10 4 /mL was inoculated into a 96-well plate, and after 24 hours of culture, the Hanshin grass drug, the composition drug 2, the composition drug 4, and the composition drug 5 were respectively added. The final concentration was equivalent to 1500 μg/mL of Hanxin herbal medicine, and a negative control group was set up. Each group of 6 parallel wells was cultured in a 37 ° C, 5% CO 2 cell incubator for 72 h, then the supernatant was centrifuged, washed with PBS, Collect cells and set aside.
5、qRT-PCR测定HeLa细胞p62基因和HO-8910人卵巢癌细胞LSD1基因的表达水平5, qRT-PCR determination of HeLa cell p62 gene and HO-8910 human ovarian cancer cell LSD1 gene expression level
收集上述细胞，用PBS洗涤细胞3次，按照RNAiso Plus试剂盒操作手册提取细胞总RNA，并用紫外分光光度法行定量分析。采用PrimeScript RT试剂盒将RNA反转录为cDNA，以cDNA为模板，采用SYBR-Green PCR Master Mix试剂盒进一步行PCR扩增。The above cells were collected, and the cells were washed 3 times with PBS, and total RNA was extracted according to the RNAiso Plus kit operating manual, and quantitative analysis was carried out by ultraviolet spectrophotometry. The RNA was reverse transcribed into cDNA using the PrimeScript RT kit, and cDNA amplification was carried out using the SYBR-Green PCR Master Mix kit using cDNA as a template.
p62基因上游引物序列为5’-TTTTCACTGTCAATTCACTGCA-3’；The upstream primer sequence of the p62 gene is 5'-TTTTCACTGTCAATTCACTGCA-3';
p62基因下游引物序列为5’-GGAGGCGAGGATGGAGGAA-3’；The p62 gene downstream primer sequence is 5'-GGAGGCGAGGATGGAGGAA-3';
LSD1基因上游引物序列为5’-CAAGTGTCAATTTGTTCGGG-3’；The upstream primer sequence of the LSD1 gene is 5'-CAAGTGTCAATTTGTTCGGG-3';
LSD1基因下游引物序列为5’-TTCTTTGGGCTGAGGTACTG-3’；The downstream primer sequence of the LSD1 gene is 5'-TTCTTTGGGCTGAGGTACTG-3';
内参GAPDH基因上游引物序列为5’-GCAAATTCCATGGCACCGTC-3’；The upstream primer sequence of the internal reference GAPDH gene is 5'-GCAAATTCCATGGCACCGTC-3';
内参GAPDH基因下游引物序列为5’-TCGCCCCACTTGATTTTGG-3’。The downstream primer sequence of the internal reference GAPDH gene is 5'-TCGCCCCACTTGATTTTGG-3'.
PCR反应条件：90℃30s，1个循环；95℃5s、60℃30s，共40个循环。以2 -ΔΔCt值(Ct代表循环阈值)表示p62mRNA、LSD1mRNA的相对表达量。实验重复3次。 PCR reaction conditions: 90 ° C for 30 s, 1 cycle; 95 ° C 5s, 60 ° C 30s, a total of 40 cycles. The relative expression level of p62 mRNA and LSD1 mRNA is represented by a 2- ΔΔCt value (Ct represents a cycle threshold). The experiment was repeated three times.
6、统计学方法6, statistical methods
采用Graph PadPrism 5软件进行数据的统计和分析。计量资料以x±s表示，多组间比较采用单因素方差分析，两两比较采用Tukey检验。P＜0.05为差异有统计学意义。Data statistics and analysis were performed using Graph PadPrism 5 software. The measurement data were expressed as x±s, and the comparison between groups was performed by one-way analysis of variance, and the comparison between the two groups was performed by Tukey test. P < 0.05 was considered statistically significant.
三、实验结果Third, the experimental results
1、野黄芩苷、粗毛豚草素、汉黄芩苷、白杨素、芹菜素、汉黄芩素在韩信草中的含量1. Content of wild baicalin, crude ragweed, baicalin, chrysin, apigenin and wogonin in Hanxincao
野黄芩苷、粗毛豚草素、汉黄芩苷、白杨素、芹菜素、汉黄芩素在韩信草中含量如下表。The contents of wild baicalin, crude ragivin, baicalin, chrysin, apigenin, and wogonin in Hanxin grass are shown in the following table.
化合物名称Compound name 含量(mg/g)Content (mg/g) 化合物名称Compound name 含量(mg/g)Content (mg/g)
野黄芩苷Wild baicalin 1.781.78 白杨素Chrysin 0.0310.031
粗毛豚草素Corydalis 0.0650.065 芹菜素Apigenin 0.160.16
汉黄芩苷Hanknot 0.290.29 汉黄芩素Hanaxanthin 0.130.13
2、韩信草药物及组合物对HeLa细胞p62基因的表达水平的影响2. Effect of Hanxincao drugs and compositions on the expression level of p62 gene in HeLa cells
韩信草药物、组合物药物1、组合物药物3、组合物药物5对HeLa细胞p62基因的表达水平的影响如下表所示。The effects of Hanxincao drug, composition drug 1, composition drug 3, and composition drug 5 on the expression level of p62 gene of HeLa cells are shown in the following table.
组别Group p62mRNA/GAPDHmRNAp62mRNA/GAPDH mRNA 抑制率Inhibition rate
阴性对照组Negative control group 1.001.00 //
韩信草药物(相当于韩信草药材1500μg/mL)Hanxin grass drug (equivalent to Hanxin herbal medicine 1500μg/mL) 0.560.56 44％44%
组合物药物1(相当于韩信草药材1500μg/mL)Composition drug 1 (equivalent to Hanxin herbal medicine 1500μg/mL) 0.580.58 42％42%
组合物药物3(相当于韩信草药材1500μg/mL)Composition drug 3 (equivalent to Hanxin herbal medicine 1500μg/mL) 0.850.85 15％15%
组合物药物5(相当于韩信草药材1500μg/mL)Composition drug 5 (equivalent to Hanxin herbal medicine 1500μg/mL) 0.910.91 9％9%
上述实验结果说明：The above experimental results show that:
(1)由野黄芩苷、粗毛豚草素、汉黄芩苷、白杨素和芹菜素组成的组合物基本可以等效替代韩信草对p62基因表达的抑制效应，二者强度相当；该组合物可以用于等效替代韩信草制备p62基因表达抑制剂；本领域技术人员知道，抑制p62基因表达可以有效抑制宫颈癌细 胞的增殖(RNA干扰抑制p62基因表达对宫颈癌HeLa细胞增殖的影响，蚌埠医学院学报2018年1月第43卷第1期)，因此，该组合物可以用于等效替代韩信草制备宫颈癌治疗药物；(1) The composition consisting of wild baicalin, crude ragivin, baicalin, chrysin and apigenin can substantially replace the inhibitory effect of Hanxin grass on p62 gene expression, and the strength of the two is equivalent; the composition can For the equivalent replacement of Hanxincao to prepare p62 gene expression inhibitor; those skilled in the art know that inhibition of p62 gene expression can effectively inhibit the proliferation of cervical cancer cells (RNA interference inhibits p62 gene expression on cervical cancer HeLa cell proliferation, 蚌埠 medicine Journal of the Academy of Sciences, Vol. 43, No. 1, January 2018), therefore, the composition can be used for the equivalent replacement of Hanxin grass for the preparation of cervical cancer therapeutic drugs;
(2)由野黄芩苷、粗毛豚草素、汉黄芩苷、白杨素和芹菜素组成的组合物中，粗毛豚草素对p62基因表达的独立抑制率是9％，其他4种成分对p62基因表达的独立抑制率是15％，二者之和(9％+15％＝24％)显著小于5种成分对p62基因表达的组合抑制率42％，这种1+1＜2的现象体现了该组合物中粗毛豚草素与其他4种成分存在明显的协同抑制p62基因表达的效应。(2) In the composition consisting of wild baicalin, crude ragivin, scutellarin, chrysin and apigenin, the independent inhibition rate of p62 gene expression by bursalin was 9%, and the other four components were p62. The independent inhibition rate of gene expression was 15%, and the sum of the two (9%+15%=24%) was significantly less than the combined inhibition rate of p62 gene expression by 5 components. This phenomenon of 1+1<2 is reflected. In the composition, the crude bursin and the other four components have an obvious synergistic effect of inhibiting p62 gene expression.
3、韩信草药物及组合物对HO-8910人卵巢癌细胞LSD1基因的表达水平的影响3. Effect of Hanxincao drugs and compositions on the expression level of LSD1 gene in HO-8910 human ovarian cancer cells
韩信草药物、组合物药物2、组合物药物4、组合物药物5对HO-8910人卵巢癌细胞LSD1基因的表达水平的影响如下表所示。The effects of Hanxincao drug, composition drug 2, composition drug 4, and composition drug 5 on the expression level of HO-8910 human ovarian cancer cell LSD1 gene are shown in the following table.
组别Group LSD1mRNA/GAPDHmRNALSD1 mRNA/GAPDH mRNA 抑制率Inhibition rate
阴性对照组Negative control group 1.001.00 //
韩信草药物(相当于韩信草药材1500μg/mL)Hanxin grass drug (equivalent to Hanxin herbal medicine 1500μg/mL) 0.570.57 43％43%
组合物药物2(相当于韩信草药材1500μg/mL)Composition drug 2 (equivalent to Hanxin herbal medicine 1500μg/mL) 0.550.55 45％45%
组合物药物4(相当于韩信草药材1500μg/mL)Composition drug 4 (equivalent to Hanxin herbal medicine 1500μg/mL) 0.810.81 19％19%
组合物药物5(相当于韩信草药材1500μg/mL)Composition drug 5 (equivalent to Hanxin herbal medicine 1500μg/mL) 0.900.90 10％10%
上述实验结果说明：The above experimental results show that:
(1)由野黄芩苷、粗毛豚草素、白杨素、芹菜素和汉黄芩素组成的组合物基本可以等效替代韩信草对LSD1基因表达的抑制效应，二者强度相当；该组合物可以用于等效替代韩信草制备LSD1基因表达抑制剂；本领域技术人员知道，抑制LSD1基因表达可以有效抑制卵巢癌细胞的增殖(沉默LSD1基因的表达可抑制卵巢癌HO8910细胞的增殖并诱导其凋亡，肿瘤2016年5月第36卷第5期)，因此，该组合物可以用于等效替代韩信草制备卵巢癌治疗药物；(1) A composition consisting of wild baicalin, crude ragivin, chrysin, apigenin and wogonin can substantially replace the inhibitory effect of Hanxin grass on the expression of LSD1 gene, and the strength of the two is equivalent; For the equivalent replacement of Hanxin grass to prepare LSD1 gene expression inhibitor; those skilled in the art know that inhibition of LSD1 gene expression can effectively inhibit the proliferation of ovarian cancer cells (silencing of LSD1 gene expression can inhibit the proliferation of ovarian cancer HO8910 cells and induce their withering Death, tumor, May 2016, Vol. 36, No. 5), therefore, the composition can be used for the equivalent replacement of Hanxin grass for the preparation of ovarian cancer therapeutic drugs;
(2)由野黄芩苷、粗毛豚草素、白杨素、芹菜素和汉黄芩素组成的组合物中，粗毛豚草素对LSD1基因表达的独立抑制率是10％，其他4种成分对LSD1基因表达的组合抑制率是19％，二者之和(10％+19％＝29％)显著小于5种成分对LSD1基因表达的组合抑制率45％，这种1+1＜2的现象体现了该组合物中粗毛豚草素与其他4种成分存在明显的协同抑制LSD1基因表达的效应。(2) In the composition consisting of wild baicalin, crude ragivin, chrysin, apigenin and wogonin, the independent inhibition rate of LDD1 gene expression by burrowin is 10%, and the other 4 components are to LSD1. The combined inhibition rate of gene expression was 19%, and the sum of the two (10%+19%=29%) was significantly less than the combined inhibition rate of LSD1 gene expression by 4 components. This phenomenon of 1+1<2 is reflected. In the composition, the crude bursin and the other four components have an obvious synergistic effect of inhibiting the expression of the LSD1 gene.
实施例3：苍耳子等效成分群Example 3: Xanthium equivalent component group
一、实验材料First, the experimental materials
苍耳子药材购于亳州药材市场，产地为辽宁本溪，粉碎后55℃烘干过夜，干燥保存备用。Xanthium medicinal herbs are purchased from the Chuzhou medicinal materials market. The origin is Benxi, Liaoning. After smashing, it is dried overnight at 55 °C, and dried for storage.
Agilent 1100液相色谱仪；Sartorius BS210S型电子天平，北京赛多利斯仪器系统有限公司；Sartorius CP225D型电子天平，北京赛多利斯仪器系统有限公司；乙腈(色谱纯)、甲醇(色谱纯)购自美国天地(TEDIA)；水为娃哈哈纯净水。Agilent 1100 liquid chromatograph; Sartorius BS210S electronic balance, Beijing Sartorius Instrument System Co., Ltd.; Sartorius CP225D electronic balance, Beijing Sartorius Instrument System Co., Ltd.; acetonitrile (chromatographically pure), methanol (chromatographically pure) purchased from American World (TEDIA); water is Wahaha pure water.
RNAiso plus试剂、SYBRGreen PCRMaster Mix和PrimeScript RT试剂盒购自TaKaRa。RNAiso plus reagent, SYBR Green PCR Master Mix and PrimeScript RT kit were purchased from TaKaRa.
二、实验方法Second, the experimental method
1、新绿原酸、绿原酸、隐绿原酸、噻嗪双酮苷、1,5-二咖啡酰奎宁酸、4,5-二咖啡酰奎宁酸在苍耳子中的含量测定1. Determination of new chlorogenic acid, chlorogenic acid, cryptochlorogenic acid, thiazide diketide, 1,5-dicaffeoylquinic acid and 4,5-dicaffeoylquinic acid in Xanthium sibiricum
HPLC方法包括如下色谱参数：The HPLC method includes the following chromatographic parameters:
色谱仪：Agilent 1100液相色谱仪Chromatograph: Agilent 1100 Liquid Chromatograph
色谱柱：Eclipse XDB-C18液相色谱柱(250mm×4.6mm，5μm)；Column: Eclipse XDB-C18 liquid chromatography column (250mm × 4.6mm, 5μm);
流动相：甲醇(A)-0.1％磷酸水溶液(B)；Mobile phase: methanol (A) - 0.1% aqueous phosphoric acid solution (B);
洗脱程序：0min，10％A；5min，15％A；12min，15％A；15min，20％A；20min，25％A；25min，25％A；30min，27％A；32min，35％A；60min，40％A；Elution procedure: 0 min, 10% A; 5 min, 15% A; 12 min, 15% A; 15 min, 20% A; 20 min, 25% A; 25 min, 25% A; 30 min, 27% A; 32 min, 35% A; 60 min, 40% A;
柱温：35℃。Column temperature: 35 ° C.
检测波长：254nm。Detection wavelength: 254 nm.
流动相流速：1.0mL/min。Mobile phase flow rate: 1.0 mL/min.
进样量：20μL。Injection volume: 20 μL.
供试品溶液的制备：将苍耳子药材粉碎，过40目筛，精密称取10g，置圆底烧瓶中，加入70％乙醇40mL，水浴回流2h，滤过，取滤液，重复3次。合并滤液，蒸去乙醇后干燥，得粉末。将粉末溶于250mL甲醇，过微孔滤膜，即得。Preparation of the test solution: The scorpion medicinal material was pulverized, passed through a 40 mesh sieve, and accurately weighed 10 g, placed in a round bottom flask, added with 40% ethanol 40 mL, refluxed in a water bath for 2 h, filtered, and the filtrate was taken and repeated three times. The filtrate was combined, evaporated to ethanol and dried to give a powder. The powder was dissolved in 250 mL of methanol and passed through a microporous membrane to obtain.
精密量取供试品溶液20μL注入液相色谱仪，用标准品外标法测定新绿原酸、绿原酸、隐绿原酸、噻嗪双酮苷、1,5-二咖啡酰奎宁酸、4,5-二咖啡酰奎宁酸在苍耳子中的含量。Precisely take 20 μL of the test solution into the liquid chromatograph and measure the new chlorogenic acid, chlorogenic acid, cryptochlorogenic acid, thiazinidine, and 1,5-dicaffeoylquinic acid by standard external standard method. And the content of 4,5-dicaffeoylquinic acid in Xanthium.
2、细胞培养2, cell culture
人前列腺癌PC-3细胞用含10％胎牛血清的F12培养基于37℃、5％CO 2条件下传代培养。脑胶质瘤U251细胞用含10％胎牛血清的DMEM培养基于37℃、5％CO 2条件下传代培养。取对数生长期细胞用于实验。 Human prostate cancer PC-3 cells were subcultured in F12 medium containing 10% fetal calf serum at 37 ° C under 5% CO 2 . Glioblastoma U251 cells were subcultured in DMEM medium containing 10% fetal calf serum at 37 ° C under 5% CO 2 . Logarithmic growth phase cells were used for experiments.
3、药物制备3, drug preparation
苍耳子药物：将苍耳子药材粉碎，过40目筛，精密称取10g，置圆底烧瓶中，加入70％乙醇40mL，水浴回流2h，滤过，取滤液，重复3次。合并滤液，蒸去乙醇后干燥，得粉末。Xanthium drug: The scorpion medicinal material is crushed, passed through a 40 mesh sieve, accurately weighed 10 g, placed in a round bottom flask, added 40 mL of 70% ethanol, refluxed for 2 h in a water bath, filtered, and the filtrate was taken and repeated 3 times. The filtrate was combined, evaporated to ethanol and dried to give a powder.
组合物药物所含成分种类如下表，各成分按照在苍耳子的含量比例用标准品勾兑制得。The components of the composition drug are listed in the following table, and each component is prepared by blending with a standard product according to the content ratio of Xanthium.
Figure PCTCN2018103855-appb-000001
Figure PCTCN2018103855-appb-000001
4、分组及给药4, grouping and drug delivery
将浓度为5×10 4/mL的人前列腺癌PC-3细胞悬液接种于96孔板中，培养24h后，分别加入苍耳子药物、组合物药物1、组合物药物3、组合物药物5使其终浓度相当于苍耳子药材2500μg/mL，同时设阴性对照组，每组6个平行孔，37℃、5％CO 2细胞培养箱中培养72h后，离心弃上清液，PBS洗涤、收集细胞，备用。 The human prostate cancer PC-3 cell suspension at a concentration of 5×10 4 /mL was inoculated into a 96-well plate, and after 24 hours of culture, the Xanthium drug, the composition drug 1, the composition drug 3, and the composition drug were respectively added. 5 The final concentration was equivalent to 2500 μg/mL of Xanthium sibiricum, and a negative control group was set up. Each group of 6 parallel wells was cultured in a 37 ° C, 5% CO 2 cell incubator for 72 h, then centrifuged to discard the supernatant, PBS. Wash, collect cells, and set aside.
将浓度为5×10 4/mL的脑胶质瘤U251细胞悬液接种于96孔板中，培养24h后，分别加入苍耳子药物、组合物药物2、组合物药物4、组合物药物5使其终浓度相当于苍耳子药材2500μg/mL，同时设阴性对照组，每组6个平行孔，37℃、5％CO 2细胞培养箱中培养72h后，离心弃上清液，PBS洗涤、收集细胞，备用。 The glioma U251 cell suspension at a concentration of 5×10 4 /mL was inoculated into a 96-well plate, and after 24 hours of culture, the Xanthium drug, the composition drug 2, the composition drug 4, and the composition drug 5 were respectively added. The final concentration was equivalent to 2500 μg/mL of Xanthium sibiricum, and the negative control group was set up. Each group of 6 parallel wells was cultured in a 37 ° C, 5% CO 2 cell incubator for 72 h, then the supernatant was centrifuged and washed with PBS. Collect cells and set aside.
5、qRT-PCR测定人前列腺癌PC-3细胞中AMPK基因和脑胶质瘤U251细胞中mTOR基因的表达水平5. qRT-PCR was used to detect the expression level of mTOR gene in AMPK gene and glioma U251 cells in human prostate cancer PC-3 cells.
收集上述细胞，用PBS洗涤细胞3次，按照RNAiso Plus试剂盒操作手册提取细胞总RNA，并用紫外分光光度法行定量分析。采用PrimeScript RT试剂盒将RNA反转录为cDNA，以cDNA为模板，采用SYBR-Green PCR Master Mix试剂盒进一步行PCR扩增。The above cells were collected, and the cells were washed 3 times with PBS, and total RNA was extracted according to the RNAiso Plus kit operating manual, and quantitative analysis was carried out by ultraviolet spectrophotometry. The RNA was reverse transcribed into cDNA using the PrimeScript RT kit, and cDNA amplification was carried out using the SYBR-Green PCR Master Mix kit using cDNA as a template.
AMPK基因上游引物序列为5’-TTGGTCAATGAAACCACTATCTG-3’；The upstream primer sequence of the AMPK gene is 5'-TTGGTCAATGAAACCACTATCTG-3';
AMPK基因下游引物序列为5’-CGTTCGGCCATGATGTGTTAAGT-3’；The downstream primer sequence of the AMPK gene is 5'-CGTTCGGCCATGATGTGTTAAGT-3';
mTOR基因上游引物序列为5’-GCGAACCTCAGGGCAAGAT-3’；The upstream primer sequence of the mTOR gene is 5'-GCGAACCTCAGGGCAAGAT-3';
mTOR基因下游引物序列为5’-TGACTCATCTCTCGGAGTTCCA-3’；The downstream primer sequence of the mTOR gene is 5'-TGACTCATCTCTCGGAGTTCCA-3';
内参GAPDH基因上游引物序列为5’-GCAAATTCCATGGCACCGTC-3’；The upstream primer sequence of the internal reference GAPDH gene is 5'-GCAAATTCCATGGCACCGTC-3';
内参GAPDH基因下游引物序列为5’-TCGCCCCACTTGATTTTGG-3’。The downstream primer sequence of the internal reference GAPDH gene is 5'-TCGCCCCACTTGATTTTGG-3'.
PCR反应条件：90℃30s，1个循环；95℃5s、60℃30s，共40个循环。以2 -ΔΔCt值(Ct代表循环阈值)表示AMPKmRNA、mTOR mRNA的相对表达量。实验重复3次。 PCR reaction conditions: 90 ° C for 30 s, 1 cycle; 95 ° C 5s, 60 ° C 30s, a total of 40 cycles. The relative expression level of AMPK mRNA and mTOR mRNA is represented by a 2- ΔΔCt value (Ct represents a cycle threshold). The experiment was repeated three times.
6、统计学方法6, statistical methods
采用Graph PadPrism 5软件进行数据的统计和分析。计量资料以x±s表示，多组间比较采用单因素方差分析，两两比较采用Tukey检验。P＜0.05为差异有统计学意义。Data statistics and analysis were performed using Graph PadPrism 5 software. The measurement data were expressed as x±s, and the comparison between groups was performed by one-way analysis of variance, and the comparison between the two groups was performed by Tukey test. P < 0.05 was considered statistically significant.
三、实验结果Third, the experimental results
1、新绿原酸、绿原酸、隐绿原酸、噻嗪双酮苷、1,5-二咖啡酰奎宁酸、4,5-二咖啡酰奎宁酸在苍耳子中的含量1. Content of new chlorogenic acid, chlorogenic acid, cryptochlorogenic acid, thiazide diketide, 1,5-dicaffeoylquinic acid and 4,5-dicaffeoylquinic acid in Xanthium sibiricum
新绿原酸、绿原酸、隐绿原酸、噻嗪双酮苷、1,5-二咖啡酰奎宁酸、4,5-二咖啡酰奎宁酸在苍耳子中含量如下表。The contents of new chlorogenic acid, chlorogenic acid, cryptochlorogenic acid, thiazide diketide, 1,5-dicaffeoylquinic acid and 4,5-dicaffeoylquinic acid in Xanthium are shown in the following table.
化合物名称Compound name 含量(mg/g)Content (mg/g) 化合物名称Compound name 含量(mg/g)Content (mg/g)
新绿原酸New chlorogenic acid 0.220.22 噻嗪双酮苷Thiazinium diketide 0.730.73
绿原酸Chlorogenic acid 0.960.96 1,5-二咖啡酰奎宁酸1,5-dicaffeoylquinic acid 1.341.34
隐绿原酸Chlorogenic acid 0.670.67 4,5-二咖啡酰奎宁酸4,5-dicaffeoylquinic acid 0.530.53
2、苍耳子药物及组合物对人前列腺癌PC-3细胞AMPK基因的表达水平的影响2. Effect of Xanthium sibiricum and its composition on the expression level of AMPK gene in human prostate cancer PC-3 cells
苍耳子药物、组合物药物1、组合物药物3、组合物药物5对人前列腺癌PC-3细胞AMPK基因的表达水平的影响如下表所示。The effects of Xanthium drug, composition drug 1, composition drug 3, and composition drug 5 on the expression level of AMPK gene in human prostate cancer PC-3 cells are shown in the following table.
组别Group AMPKmRNA/GAPDHmRNAAMPK mRNA/GAPDH mRNA 抑制率Inhibition rate
阴性对照组Negative control group 1.001.00 //
苍耳子药物(相当于苍耳子药材2500μg/mL)Xanthium drug (equivalent to 2500μg/mL of Xanthium sibiricum) 0.410.41 59％59%
组合物药物1(相当于苍耳子药材2500μg/mL)Composition drug 1 (equivalent to 2500μg/mL of Xanthium sibiricum) 0.450.45 55％55%
组合物药物3(相当于苍耳子药材2500μg/mL)Composition drug 3 (equivalent to 2500μg/mL of Xanthium sibiricum) 0.790.79 21％twenty one%
组合物药物5(相当于苍耳子药材2500μg/mL)Composition drug 5 (equivalent to 2500μg/mL of Xanthium sibiricum) 0.920.92 8％8%
上述实验结果说明：The above experimental results show that:
(1)由新绿原酸、隐绿原酸、噻嗪双酮苷、1,5-二咖啡酰奎宁酸、4,5-二咖啡酰奎宁酸组成的组合物基本可以等效替代苍耳子对AMPK基因表达的抑制效应，二者强度相当；该组合物可以用于等效替代苍耳子制备AMPK基因表达抑制剂；本领域技术人员知道，抑制AMPK基因表达可以有效抑制前列腺癌细胞的增殖(RNA干扰沉默AMPK对前列腺癌细胞生物学行为影响，中华肿瘤防治杂志2018年1月第25卷第2期)，因此，该组合物可以用于等效替代苍耳子制备前列腺癌治疗药物；(1) A composition consisting of neochlorogenic acid, cryptochlorogenic acid, thiazinidine, 1,5-dicaffeoylquinic acid, 4,5-dicaffeoylquinic acid can be substantially substituted for Cang The inhibitory effect of the ear on the expression of AMPK gene is equivalent; the composition can be used to prepare an inhibitor of AMPK gene expression by equivalent substitution of Xanthium; those skilled in the art know that inhibition of AMPK gene expression can effectively inhibit prostate cancer cells Proliferation (RNA interference silencing AMPK on the biological behavior of prostate cancer cells, Chinese Journal of Cancer Prevention and Treatment, Vol. 25, No. 2, January 2018), therefore, the composition can be used for the equivalent replacement of Xanthium in the preparation of prostate cancer drug;
(2)由新绿原酸、隐绿原酸、噻嗪双酮苷、1,5-二咖啡酰奎宁酸、4,5-二咖啡酰奎宁酸组成的组合物中，噻嗪双酮苷对AMPK基因表达的独立抑制率是8％，其他4种成分对AMPK基因表达的独立抑制率是21％，二者之和(8％+21％＝29％)显著小于5种成分对AMPK基因表达的组合抑制率55％，这种1+1＜2的现象体现了该组合物中噻嗪双酮苷与其他4种成分存 在明显的协同抑制AMPK基因表达的效应。(2) a thiazide dione in a composition consisting of neochlorogenic acid, cryptochlorogenic acid, thiazide diketide, 1,5-dicaffeoylquinic acid, 4,5-dicaffeoylquinic acid The independent inhibition rate of AMPK gene expression by glucoside was 8%, and the independent inhibition rate of AMPK gene expression by the other four components was 21%, and the sum of the two (8%+21%=29%) was significantly less than 5 components to AMPK. The combined inhibition rate of gene expression is 55%. This phenomenon of 1+1<2 reflects the effect of the thiazide diketide in the composition and the other four components on the synergistic inhibition of AMPK gene expression.
3、苍耳子药物及组合物对脑胶质瘤U251细胞mTOR基因的表达水平的影响Effects of Xanthium drugs and compositions on the expression of mTOR gene in glioma U251 cells
苍耳子药物、组合物药物2、组合物药物4、组合物药物5对脑胶质瘤U251细胞mTOR基因的表达水平的影响如下表所示。The effects of Xanthium drug, composition drug 2, composition drug 4, and composition drug 5 on the expression level of mTOR gene in glioma U251 cells are shown in the following table.
组别Group mTORmRNA/GAPDHmRNAmTORmRNA/GAPDHmRNA 抑制率Inhibition rate
阴性对照组Negative control group 1.001.00 //
苍耳子药物(相当于苍耳子药材2500μg/mL)Xanthium drug (equivalent to 2500μg/mL of Xanthium sibiricum) 0.450.45 55％55%
组合物药物2(相当于苍耳子药材2500μg/mL)Composition drug 2 (equivalent to 2500μg/mL of Xanthium sibiricum) 0.430.43 57％57%
组合物药物4(相当于苍耳子药材2500μg/mL)Composition drug 4 (equivalent to 2500μg/mL of Xanthium sibiricum) 0.760.76 24％twenty four%
组合物药物5(相当于苍耳子药材2500μg/mL)Composition drug 5 (equivalent to 2500μg/mL of Xanthium sibiricum) 0.870.87 13％13%
上述实验结果说明：The above experimental results show that:
(1)由新绿原酸、绿原酸、噻嗪双酮苷、1,5-二咖啡酰奎宁酸、4,5-二咖啡酰奎宁酸组成的组合物基本可以等效替代苍耳子对mTOR基因表达的抑制效应，二者强度相当；该组合物可以用于等效替代苍耳子制备mTOR基因表达抑制剂；本领域技术人员知道，抑制mTOR基因表达可以有效抑制脑胶质瘤细胞的增殖(miRNA-99b负调控mTOR抑制脑胶质瘤细胞的侵袭能力，中国药理学通报2018年4月第34卷第4期)，因此，该组合物可以用于等效替代苍耳子制备脑胶质瘤治疗药物；(1) A composition consisting of neochlorogenic acid, chlorogenic acid, thiazinidine, 1,5-dicaffeylquinic acid, 4,5-dicaffeoylquinic acid can be substantially substituted for Xanthium The inhibitory effect of the sub-mTOR gene expression is equivalent to each other; the composition can be used for the equivalent replacement of Xanthium for the preparation of mTOR gene expression inhibitor; those skilled in the art know that inhibition of mTOR gene expression can effectively inhibit glioma Cell proliferation (miRNA-99b negatively regulates mTOR inhibition of glioma cell invasion, Chinese Journal of Pharmacology, April 2018, Vol. 34, No. 4), therefore, the composition can be used for equivalent replacement of Xanthium Preparation of glioma therapeutic drugs;
(2)由新绿原酸、绿原酸、噻嗪双酮苷、1,5-二咖啡酰奎宁酸、4,5-二咖啡酰奎宁酸组成的组合物中，噻嗪双酮苷对mTOR基因表达的独立抑制率是13％，其他4种成分对mTOR基因表达的组合抑制率是24％，二者之和(13％+24％＝37％)显著小于5种成分对mTOR基因表达的组合抑制率57％，这种1+1＜2的现象体现了该组合物中噻嗪双酮苷与其他4种成分存在明显的协同抑制mTOR基因表达的效应。(2) a thiazide diketide in a composition consisting of neochlorogenic acid, chlorogenic acid, thiazinidine, 1,5-dicaffeoylquinic acid, 4,5-dicaffeoylquinic acid The independent inhibition rate of mTOR gene expression was 13%, and the combined inhibition rate of mTOR gene expression by the other four components was 24%, and the sum of the two (13%+24%=37%) was significantly smaller than the five components of the mTOR gene. The combined inhibition rate of expression was 57%. This 1+1<2 phenomenon embodies the effect of the synergistic inhibition of mTOR gene expression by the thiazindione glycoside and the other four components in the composition.
实施例4：锥叶柴胡等效成分群Example 4: Equivalent component group of Bupleurum chinense
一、实验材料First, the experimental materials
锥叶柴胡药材收集自内蒙古海拉尔，粉碎后55℃烘干过夜，干燥保存备用。The medicinal material of B. chinensis was collected from Hailar, Inner Mongolia, dried at 55 ° C overnight, and dried for storage.
Agilent 1100液相色谱仪；Sartorius BS210S型电子天平，北京赛多利斯仪器系统有限公司；Sartorius CP225D型电子天平，北京赛多利斯仪器系统有限公司；乙腈(色谱纯)、甲醇(色谱纯)购自美国天地(TEDIA)；水为娃哈哈纯净水。Agilent 1100 liquid chromatograph; Sartorius BS210S electronic balance, Beijing Sartorius Instrument System Co., Ltd.; Sartorius CP225D electronic balance, Beijing Sartorius Instrument System Co., Ltd.; acetonitrile (chromatographically pure), methanol (chromatographically pure) purchased from American World (TEDIA); water is Wahaha pure water.
RNAiso plus试剂、SYBRGreen PCRMaster Mix和PrimeScript RT试剂盒购自TaKaRa。RNAiso plus reagent, SYBR Green PCR Master Mix and PrimeScript RT kit were purchased from TaKaRa.
二、实验方法Second, the experimental method
1、柴胡次皂苷F、柴胡次皂苷G、柴胡次皂苷I、柴胡皂苷C、柴胡皂苷A、柴胡皂苷D 在锥叶柴胡中的含量测定1. Determination of the content of saikosaponin F, saikosaponin G, saikosaponin I, saikosaponin C, saikosaponin A and saikosaponin D in cone Bupleurum
HPLC方法包括如下色谱参数：The HPLC method includes the following chromatographic parameters:
色谱仪：Agilent 1100液相色谱仪Chromatograph: Agilent 1100 Liquid Chromatograph
色谱柱：InertSustainAQ-C18液相色谱柱(250mm×4.6mm，5μm)；Column: InertSustain AQ-C18 liquid chromatography column (250 mm × 4.6 mm, 5 μm);
流动相：乙腈(A)-水(B)；Mobile phase: acetonitrile (A) - water (B);
洗脱程序：0～10min，20％→30％A；10～20min，30％→40％A；20～45min，40％→60％A；45～55min，60％→90％A；Elution procedure: 0 ~ 10min, 20% → 30% A; 10 ~ 20min, 30% → 40% A; 20 ~ 45min, 40% → 60% A; 45 ~ 55min, 60% → 90% A;
柱温：25℃。Column temperature: 25 ° C.
检测波长：210nm。Detection wavelength: 210 nm.
流动相流速：1.0mL/min。Mobile phase flow rate: 1.0 mL/min.
进样量：20μL。Injection volume: 20 μL.
供试品溶液的制备：将锥叶柴胡药材粉碎，过40目筛，精密称取10g，置圆底烧瓶中，加入70％乙醇40mL，水浴回流2h，滤过，取滤液，重复3次。合并滤液，蒸去乙醇后干燥，得粉末。将粉末溶于250mL甲醇，过微孔滤膜，即得。Preparation of the test solution: The stalked Bupleurum chinense was pulverized, passed through a 40 mesh sieve, accurately weighed 10 g, placed in a round bottom flask, added with 40% ethanol 40 mL, refluxed in a water bath for 2 h, filtered, and the filtrate was taken, repeated 3 times. . The filtrate was combined, evaporated to ethanol and dried to give a powder. The powder was dissolved in 250 mL of methanol and passed through a microporous membrane to obtain.
精密量取供试品溶液20μL注入液相色谱仪，用标准品外标法测定柴胡次皂苷F、柴胡次皂苷G、柴胡次皂苷I、柴胡皂苷C、柴胡皂苷A、柴胡皂苷D在锥叶柴胡中的含量。Precisely measure 20 μL of the test solution into the liquid chromatograph, and determine the saponin F, Chae saponin G, Chae saponin I, saikosaponin C, saikosaponin A, and firewood using standard external standard method. The content of saponin D in Bupleurum chinense.
2、细胞培养2, cell culture
人前列腺癌PC-3细胞用含10％胎牛血清的F12培养基于37℃、5％CO 2条件下传代培养。脑胶质瘤U251细胞用含10％胎牛血清的DMEM培养基于37℃、5％CO 2条件下传代培养。取对数生长期细胞用于实验。 Human prostate cancer PC-3 cells were subcultured in F12 medium containing 10% fetal calf serum at 37 ° C under 5% CO 2 . Glioblastoma U251 cells were subcultured in DMEM medium containing 10% fetal calf serum at 37 ° C under 5% CO 2 . Logarithmic growth phase cells were used for experiments.
3、药物制备3, drug preparation
锥叶柴胡药物：将锥叶柴胡药材粉碎，过40目筛，精密称取10g，置圆底烧瓶中，加入70％乙醇40mL，水浴回流2h，滤过，取滤液，重复3次。合并滤液，蒸去乙醇后干燥，得粉末。Cone Bupleurum medicine: The medicinal material of B. chinensis was crushed, passed through a 40 mesh sieve, and accurately weighed 10 g, placed in a round bottom flask, added with 40 mL of ethanol 40 mL, refluxed for 2 h in a water bath, filtered, and the filtrate was taken and repeated 3 times. The filtrate was combined, evaporated to ethanol and dried to give a powder.
组合物药物所含成分种类如下表，各成分按照在锥叶柴胡的含量比例用标准品勾兑制得。The components of the composition drug are listed in the following table, and each component is prepared by blending with a standard product according to the content ratio of Bupleurum chinense.
序号Serial number 数量Quantity 所含成分种类Type of ingredients
组合物药物1Composition drug 1 55 柴胡次皂苷F、柴胡次皂苷I、柴胡皂苷C、柴胡皂苷A、柴胡皂苷DBupleurum saponin F, saikosaponin I, saikosaponin C, saikosaponin A, saikosaponin D
组合物药物2Composition drug 2 55 柴胡次皂苷F、柴胡次皂苷G、柴胡皂苷C、柴胡皂苷A、柴胡皂苷DSaikosaponin F, Chae saponin G, saikosaponin C, saikosaponin A, saikosaponin D
组合物药物3Composition drug 3 44 柴胡次皂苷F、柴胡次皂苷I、柴胡皂苷A、柴胡皂苷DSaikosaponin F, Chae saponin I, saikosaponin A, saikosaponin D
组合物药物4Composition drug 4 44 柴胡次皂苷F、柴胡次皂苷G、柴胡皂苷A、柴胡皂苷DSaikosaponin F, Chae saponin G, saikosaponin A, saikosaponin D
组合物药物5Composition drug 5 11 柴胡皂苷CSaikosaponin C
4、分组及给药4, grouping and drug delivery
将浓度为5×10 4/mL的人前列腺癌PC-3细胞悬液接种于96孔板中，培养24h后，分别加入锥叶柴胡药物、组合物药物1、组合物药物3、组合物药物5使其终浓度相当于锥叶柴胡药材400μg/mL，同时设阴性对照组，每组6个平行孔，37℃、5％CO 2细胞培养箱中培养48h后，离心弃上清液，PBS洗涤、收集细胞，备用。 The human prostate cancer PC-3 cell suspension at a concentration of 5×10 4 /mL was inoculated into a 96-well plate, and after 24 hours of culture, the coniferous Bupleurum drug, the composition drug 1, the composition drug 3, and the composition were respectively added. Drug 5 was made to have a final concentration equivalent to 400 μg/mL of B. chinensis, and a negative control group was set up. Each group of 6 parallel wells was cultured in a 37 ° C, 5% CO 2 cell incubator for 48 h, and the supernatant was centrifuged. Wash with PBS, collect the cells, and set aside.
将浓度为5×10 4/mL的脑胶质瘤U251细胞悬液接种于96孔板中，培养24h后，分别加入锥叶柴胡药物、组合物药物2、组合物药物4、组合物药物5使其终浓度相当于锥叶柴胡药材400μg/mL，同时设阴性对照组，每组6个平行孔，37℃、5％CO 2细胞培养箱中培养48h后，离心弃上清液，PBS洗涤、收集细胞，备用。 The glioma U251 cell suspension at a concentration of 5×10 4 /mL was inoculated into a 96-well plate, and after 24 hours of culture, the coniferous Bupleurum drug, the composition drug 2, the composition drug 4, and the composition drug were respectively added. 5 The final concentration was equivalent to 400 μg/mL of B. chinensis medicinal herbs, and a negative control group was set up. Each group of 6 parallel wells was cultured for 48 hours in a 37 ° C, 5% CO 2 cell incubator, and the supernatant was centrifuged. The cells were washed with PBS, and the cells were collected and used.
5、qRT-PCR测定人前列腺癌PC-3细胞中AMPK基因和脑胶质瘤U251细胞中mTOR基因的表达水平5. qRT-PCR was used to detect the expression level of mTOR gene in AMPK gene and glioma U251 cells in human prostate cancer PC-3 cells.
收集上述细胞，用PBS洗涤细胞3次，按照RNAiso Plus试剂盒操作手册提取细胞总RNA，并用紫外分光光度法行定量分析。采用PrimeScript RT试剂盒将RNA反转录为cDNA，以cDNA为模板，采用SYBR-Green PCR Master Mix试剂盒进一步行PCR扩增。The above cells were collected, and the cells were washed 3 times with PBS, and total RNA was extracted according to the RNAiso Plus kit operating manual, and quantitative analysis was carried out by ultraviolet spectrophotometry. The RNA was reverse transcribed into cDNA using the PrimeScript RT kit, and cDNA amplification was carried out using the SYBR-Green PCR Master Mix kit using cDNA as a template.
AMPK基因上游引物序列为5’-TTGGTCAATGAAACCACTATCTG-3’；The upstream primer sequence of the AMPK gene is 5'-TTGGTCAATGAAACCACTATCTG-3';
AMPK基因下游引物序列为5’-CGTTCGGCCATGATGTGTTAAGT-3’；The downstream primer sequence of the AMPK gene is 5'-CGTTCGGCCATGATGTGTTAAGT-3';
mTOR基因上游引物序列为5’-GCGAACCTCAGGGCAAGAT-3’；The upstream primer sequence of the mTOR gene is 5'-GCGAACCTCAGGGCAAGAT-3';
mTOR基因下游引物序列为5’-TGACTCATCTCTCGGAGTTCCA-3’；The downstream primer sequence of the mTOR gene is 5'-TGACTCATCTCTCGGAGTTCCA-3';
内参GAPDH基因上游引物序列为5’-GCAAATTCCATGGCACCGTC-3’；The upstream primer sequence of the internal reference GAPDH gene is 5'-GCAAATTCCATGGCACCGTC-3';
内参GAPDH基因下游引物序列为5’-TCGCCCCACTTGATTTTGG-3’。The downstream primer sequence of the internal reference GAPDH gene is 5'-TCGCCCCACTTGATTTTGG-3'.
PCR反应条件：90℃30s，1个循环；95℃5s、60℃30s，共40个循环。以2 -ΔΔCt值(Ct代表循环阈值)表示AMPKmRNA、mTOR mRNA的相对表达量。实验重复3次。 PCR reaction conditions: 90 ° C for 30 s, 1 cycle; 95 ° C 5s, 60 ° C 30s, a total of 40 cycles. The relative expression level of AMPK mRNA and mTOR mRNA is represented by a 2- ΔΔCt value (Ct represents a cycle threshold). The experiment was repeated three times.
6、统计学方法6, statistical methods
采用Graph PadPrism 5软件进行数据的统计和分析。计量资料以x±s表示，多组间比较采用单因素方差分析，两两比较采用Tukey检验。P＜0.05为差异有统计学意义。Data statistics and analysis were performed using Graph PadPrism 5 software. The measurement data were expressed as x±s, and the comparison between groups was performed by one-way analysis of variance, and the comparison between the two groups was performed by Tukey test. P < 0.05 was considered statistically significant.
三、实验结果Third, the experimental results
1、柴胡次皂苷F、柴胡次皂苷G、柴胡次皂苷I、柴胡皂苷C、柴胡皂苷A、柴胡皂苷D在锥叶柴胡中的含量1. Content of saikosaponin F, saikosaponin G, saikosaponin I, saikosaponin C, saikosaponin A and saikosaponin D in cone Bupleurum
柴胡次皂苷F、柴胡次皂苷G、柴胡次皂苷I、柴胡皂苷C、柴胡皂苷A、柴胡皂苷D在锥叶柴胡中含量如下表。The contents of saikosaponin F, saikosaponin G, saikosaponin I, saikosaponin C, saikosaponin A and saikosaponin D in the cone Bupleurum are as follows.
化合物名称Compound name 含量(mg/g)Content (mg/g) 化合物名称Compound name 含量(mg/g)Content (mg/g)
柴胡次皂苷FChae saponin F 8.838.83 柴胡皂苷CSaikosaponin C 2.552.55
柴胡次皂苷GChae saponin G 13.9513.95 柴胡皂苷ASaikosaponin A 3.763.76
柴胡次皂苷IChaeminosaponin I 4.684.68 柴胡皂苷DSaikosaponin D 17.5017.50
2、锥叶柴胡药物及组合物对人前列腺癌PC-3细胞AMPK基因的表达水平的影响2. Effect of Coniferous Bupleurum Drugs and Compositions on the Expression of AMPK Gene in Human Prostate Cancer PC-3 Cells
锥叶柴胡药物、组合物药物1、组合物药物3、组合物药物5对人前列腺癌PC-3细胞AMPK基因的表达水平的影响如下表所示。The effects of the cone-leaf Bupleurum drug, the composition drug 1, the composition drug 3, and the composition drug 5 on the expression level of the AMPK gene of human prostate cancer PC-3 cells are shown in the following table.
Figure PCTCN2018103855-appb-000002
Figure PCTCN2018103855-appb-000002
上述实验结果说明：The above experimental results show that:
(1)由柴胡次皂苷F、柴胡次皂苷I、柴胡皂苷C、柴胡皂苷A、柴胡皂苷D组成的组合物基本可以等效替代锥叶柴胡对AMPK基因表达的抑制效应，二者强度相当；该组合物可以用于等效替代锥叶柴胡制备AMPK基因表达抑制剂；本领域技术人员知道，抑制AMPK基因表达可以有效抑制前列腺癌细胞的增殖(RNA干扰沉默AMPK对前列腺癌细胞生物学行为影响，中华肿瘤防治杂志2018年1月第25卷第2期)，因此，该组合物可以用于等效替代锥叶柴胡制备前列腺癌治疗药物；(1) The composition consisting of saikosaponin F, saikosaponin I, saikosaponin C, saikosaponin A, saikosaponin D can basically replace the inhibitory effect of Bupleurum chinense on AMPK gene expression. The composition is equivalent in strength; the composition can be used to prepare an AMPK gene expression inhibitor equivalently to replace B. chinensis; those skilled in the art know that inhibition of AMPK gene expression can effectively inhibit proliferation of prostate cancer cells (RNA interference silencing AMPK pair) The biological behavior of prostate cancer cells, Chinese Journal of Cancer Prevention and Treatment, Vol. 25, No. 2, January 2018, therefore, the composition can be used for the equivalent replacement of Bupleurum chinense to prepare prostate cancer therapeutic drugs;
(2)由柴胡次皂苷F、柴胡次皂苷I、柴胡皂苷C、柴胡皂苷A、柴胡皂苷D组成的组合物中，柴胡皂苷C对AMPK基因表达的独立抑制率是12％，其他4种成分对AMPK基因表达的组合抑制率是23％，二者之和(12％+23％＝35％)显著小于5种成分对AMPK基因表达的组合抑制率54％，这种1+1＜2的现象体现了该组合物中柴胡皂苷C与其他4种成分存在明显的协同抑制AMPK基因表达的效应。(2) In the composition consisting of saikosaponin F, saikosaponin I, saikosaponin C, saikosaponin A, saikosaponin D, the independent inhibition rate of saikosaponin C on AMPK gene expression is 12 %, the combined inhibition rate of AMPK gene expression by the other four components was 23%, and the sum of the two (12%+23%=35%) was significantly less than the combined inhibition rate of AMPK gene expression by 54 components. The phenomenon of 1+1<2 embodies the effect of the saikosaponin C and the other four components in the composition synergistically inhibiting the expression of the AMPK gene.
3、锥叶柴胡药物及组合物对脑胶质瘤U251细胞mTOR基因的表达水平的影响Effects of Coniferous Bupleurum Drugs and Compositions on the Expression of mTOR Gene in Glioblastoma U251 Cells
锥叶柴胡药物、组合物药物2、组合物药物4、组合物药物5对脑胶质瘤U251细胞mTOR基因的表达水平的影响如下表所示。The effects of the cone leaf Bupleurum drug, the composition drug 2, the composition drug 4, and the composition drug 5 on the expression level of the mTOR gene of glioma U251 cells are shown in the following table.
Figure PCTCN2018103855-appb-000003
Figure PCTCN2018103855-appb-000003
上述实验结果说明：The above experimental results show that:
(1)由柴胡次皂苷F、柴胡次皂苷G、柴胡皂苷C、柴胡皂苷A、柴胡皂苷D组成的组合物基本可以等效替代锥叶柴胡对mTOR基因表达的抑制效应，二者强度相当；该组合物可以用于等效替代锥叶柴胡制备mTOR基因表达抑制剂；本领域技术人员知道，抑制mTOR基因表达可以有效抑制脑胶质瘤细胞的增殖(miRNA-99b负调控mTOR抑制脑胶质瘤细胞的侵袭能力，中国药理学通报2018年4月第34卷第4期)，因此，该组合物可以用于等效替代锥叶柴胡制备脑胶质瘤治疗药物；(1) The composition consisting of saikosaponin F, saikosaponin G, saikosaponin C, saikosaponin A, saikosaponin D can basically replace the inhibitory effect of camphor bupleurum on mTOR gene expression. The composition is equivalent in strength; the composition can be used to prepare an mTOR gene expression inhibitor equivalently to replace B. chinensis; those skilled in the art know that inhibition of mTOR gene expression can effectively inhibit the proliferation of glioma cells (miRNA-99b) Negative regulation of mTOR inhibits the invasive ability of glioma cells, Chinese Journal of Pharmacology, April 2018, Vol. 34, No. 4). Therefore, the composition can be used for the equivalent replacement of B. chinensis for the preparation of glioma. drug;
(2)由柴胡次皂苷F、柴胡次皂苷G、柴胡皂苷C、柴胡皂苷A、柴胡皂苷D组成的组合物中，柴胡皂苷C对mTOR基因表达的独立抑制率是11％，其他4种成分对mTOR基因表达的组合抑制率是25％，二者之和(11％+25％＝36％)显著小于5种成分对mTOR基因表达的组合抑制率58％，这种1+1＜2的现象体现了该组合物中柴胡皂苷C与其他4种成分存在明显的协同抑制mTOR基因表达的效应。(2) In the composition consisting of saikosaponin F, saikosaponin G, saikosaponin C, saikosaponin A, saikosaponin D, the independent inhibition rate of saikosaponin C on mTOR gene expression is 11 %, the combined inhibition rate of mTOR gene expression by the other four components was 25%, and the sum of the two (11%+25%=36%) was significantly less than the combined inhibition rate of the mTOR gene expression by 58 components. The phenomenon of 1+1<2 embodies the effect of the saikosaponin C and the other four components in the composition synergistically inhibiting the expression of the mTOR gene.
上述实施例的作用在于具体介绍本发明的实质性内容，但本领域技术人员应当知道，不应将本发明的保护范围局限于该具体实施例。The above embodiments are intended to specifically describe the substantial content of the present invention, but those skilled in the art should understand that the scope of the present invention should not be limited to the specific embodiments.

Claims (16)

  1. 一种用于等效替代滇重楼制备宫颈癌治疗药物的组合物，其特征在于：A composition for equivalent preparation of a drug for treating cervical cancer, which is characterized by:
    由重楼皂苷VII、偏诺皂苷-3-O-α-L-Rha(1→2)[α-L-Rha(1→4)]-β-D-Glc、重楼皂苷H、重楼皂苷II和纤细薯蓣皂苷组成。From saponin VII, saponin-3-O-α-L-Rha(1→2)[α-L-Rha(1→4)]-β-D-Glc, saponin H, heavy building Saponin II and fine diosgenin composition.
  2. 权利要求1所述的组合物在等效替代滇重楼制备宫颈癌治疗药物方面的应用。The use of the composition of claim 1 for the preparation of a medicament for the treatment of cervical cancer in an equivalent alternative to the sputum.
  3. 一种用于等效替代滇重楼制备卵巢癌治疗药物的组合物，其特征在于：A composition for equivalent preparation of ovarian cancer therapeutic medicament for ovarian cancer, characterized in that:
    由重楼皂苷VII、偏诺皂苷-3-O-α-L-Rha(1→2)[α-L-Rha(1→4)]-β-D-Glc、重楼皂苷II、纤细薯蓣皂苷和重楼皂苷I组成。From saponin VII, saponin-3-O-α-L-Rha(1→2)[α-L-Rha(1→4)]-β-D-Glc, saponin II, yam Saponin and heavy saponin I composition.
  4. 权利要求3所述的组合物在等效替代滇重楼制备卵巢癌治疗药物方面的应用。The use of the composition of claim 3 in the preparation of an ovarian cancer therapeutic agent in an equivalent replacement.
  5. 一种用于等效替代韩信草制备宫颈癌治疗药物的组合物，其特征在于：A composition for equivalent replacement of Hanxincao for the preparation of a medicament for treating cervical cancer, characterized in that:
    由野黄芩苷、粗毛豚草素、汉黄芩苷、白杨素和芹菜素组成。It is composed of wild baicalin, crude ragivin, baicalin, chrysin and apigenin.
  6. 权利要求5所述的组合物在等效替代韩信草制备宫颈癌治疗药物方面的应用。The use of the composition of claim 5 for the equivalent replacement of Hanshin grass for the preparation of a medicament for treating cervical cancer.
  7. 一种用于等效替代韩信草制备卵巢癌治疗药物的组合物，其特征在于：A composition for equivalent replacement of Hanxin grass for preparing a medicament for treating ovarian cancer, characterized in that:
    由野黄芩苷、粗毛豚草素、白杨素、芹菜素和汉黄芩素组成。It consists of wild baicalin, crude ragivin, chrysin, apigenin and wogonin.
  8. 权利要求7所述的组合物在等效替代韩信草制备卵巢癌治疗药物方面的应用。The use of the composition of claim 7 for the equivalent replacement of Hanxin grass for the preparation of a medicament for the treatment of ovarian cancer.
  9. 一种用于等效替代苍耳子制备前列腺癌治疗药物的组合物，其特征在于：A composition for equivalent preparation of a drug for treating prostate cancer, which is characterized by:
    由新绿原酸、隐绿原酸、噻嗪双酮苷、1,5-二咖啡酰奎宁酸和4,5-二咖啡酰奎宁酸组成。It consists of neochlorogenic acid, cryptochlorogenic acid, thiazide diketide, 1,5-dicaffeoylquinic acid and 4,5-dicaffeoylquinic acid.
  10. 权利要求9所述的组合物在等效替代苍耳子制备前列腺癌治疗药物方面的应用。The use of the composition of claim 9 for the equivalent replacement of Xanthium for the preparation of a medicament for the treatment of prostate cancer.
  11. 一种用于等效替代苍耳子制备脑胶质瘤治疗药物的组合物，其特征在于：A composition for the equivalent replacement of Xanthium sibiricum for preparing glioma therapeutic drugs, characterized in that:
    由新绿原酸、绿原酸、噻嗪双酮苷、1,5-二咖啡酰奎宁酸和4,5-二咖啡酰奎宁酸组成。It consists of neochlorogenic acid, chlorogenic acid, thiazide diketoside, 1,5-dicaffeoylquinic acid and 4,5-dicaffeoylquinic acid.
  12. 权利要求11所述的组合物在等效替代苍耳子制备脑胶质瘤治疗药物方面的应用。Use of the composition of claim 11 for the equivalent replacement of Xanthium for the preparation of a glioma therapeutic agent.
  13. 一种用于等效替代锥叶柴胡制备前列腺癌治疗药物的组合物，其特征在于：A composition for the equivalent replacement of Bupleurum chinense to prepare a therapeutic drug for prostate cancer, characterized in that:
    由柴胡次皂苷F、柴胡次皂苷I、柴胡皂苷C、柴胡皂苷A和柴胡皂苷D组成。It is composed of saikosaponin F, saikosaponin I, saikosaponin C, saikosaponin A and saikosaponin D.
  14. 权利要求13所述的组合物在等效替代锥叶柴胡制备前列腺癌治疗药物方面的应用。The use of the composition of claim 13 in the preparation of a medicament for the treatment of prostate cancer by an equivalent replacement of B. chinensis.
  15. 一种用于等效替代锥叶柴胡制备脑胶质瘤治疗药物的组合物，其特征在于：A composition for the equivalent replacement of Bupleurum chinense to prepare a glioma therapeutic agent, characterized in that:
    由柴胡次皂苷F、柴胡次皂苷G、柴胡皂苷C、柴胡皂苷A和柴胡皂苷D组成。It is composed of saikosaponin F, saikosaponin G, saikosaponin C, saikosaponin A and saikosaponin D.
  16. 权利要求15所述的组合物在等效替代锥叶柴胡制备脑胶质瘤治疗药物方面的应用。The use of the composition of claim 15 in the preparation of a medicament for the treatment of glioma by an equivalent replacement of B. chinensis.
PCT/CN2018/103855 2018-05-14 2018-09-03 Discovery and pharmaceutical use of equivalent component group of traditional chinese medicine rhizoma paridis yunnanensis, scutellaria indica, fructus xanthii sibirici, or bupleurum bicaule helm WO2019218543A1 (en)

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CN201810457989.7A CN108403682B (en) 2018-05-14 2018-05-14 A kind of composition and its measuring method preparing drugs for prostate cancer for substituting the achene of Siberian cocklebur
CN201810458521.XA CN108714147A (en) 2018-05-14 2018-05-14 A kind of composition and its assay method preparing glioma treatment drug for substituting the achene of Siberian cocklebur
CN201810458521.X 2018-05-14
CN201810458292.1A CN108451966A (en) 2018-05-14 2018-05-14 It is a kind of to be used to substitute composition and its assay method that paris polyphylla prepares treatment of ovarian cancer drug
CN201810457702.0A CN108721312A (en) 2018-05-14 2018-05-14 A kind of composition and its assay method preparing glioma treatment drug for substituting Radix Bupleuri bicaulis
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CN201810457702.0 2018-05-14
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CN201810458558.2 2018-05-14
CN201810457674.2A CN108714150A (en) 2018-05-14 2018-05-14 It is a kind of to be used to substitute composition and its assay method that indian skullcap herb with root prepares treatment of ovarian cancer drug
CN201810456610.0A CN108635363B (en) 2018-05-14 2018-05-14 A kind of composition and its measuring method preparing treatment of human cervical cancer drug for substituting paris polyphylla
CN201810458558.2A CN108434168A (en) 2018-05-14 2018-05-14 It is a kind of to be used to substitute composition and its assay method that Radix Bupleuri bicaulis prepares drugs for prostate cancer

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CN101693035A (en) * 2009-10-15 2010-04-14 天津大学 Medicinal preparation with inhibiting effect on tumor metastasis
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Publication number Priority date Publication date Assignee Title
CN101693035A (en) * 2009-10-15 2010-04-14 天津大学 Medicinal preparation with inhibiting effect on tumor metastasis
CN108635363A (en) * 2018-05-14 2018-10-12 杨真慧 It is a kind of to be used to substitute composition and its assay method that paris polyphylla prepares treatment of human cervical cancer drug

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