CN107913308A - A kind of Sichuan chinaberry extract and biomedical uses using root of Beijing euphorbia alkane type triterpenoid as active ingredient - Google Patents
A kind of Sichuan chinaberry extract and biomedical uses using root of Beijing euphorbia alkane type triterpenoid as active ingredient Download PDFInfo
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Abstract
The invention discloses a kind of Sichuan chinaberry extract and biomedical uses using root of Beijing euphorbia alkane type triterpenoid as active ingredient, the sum of mass percentage of Meliasenin B, C and D described in the Sichuan chinaberry extract is not less than 75%.Sichuan chinaberry extract provided by the invention can significantly inhibit the propagation of liver cancer cells by suppressing Gli1, Gli2 gene expression, and obvious dose dependent is presented in inhibitory action, the Sichuan chinaberry extract is effective inhibitor of Gli1, Gli2 gene, can be used for the medicine for preparing prevention liver cancer.One skilled in the art will appreciate that it is a kind of more mature anti-cancer molecules path to suppress Gli1, Gli2 gene expression, which is exactly based on this path and plays antihepatocarcinoma effect.It has also been found that naringenin can reduce toxicity of the Sichuan chinaberry extract of the present invention to Human normal hepatocyte, but its toxicity to liver cancer cells is not influenced.Preparation method provided by the invention is suitable for industrialized production without using silica gel column chromatography, each step, and implementation is strong.
Description
Technical field
The invention belongs to pharmaceutical technology field, extract and preparation method thereof and medical usage, and in particular to it is a kind of with
Meliasenin B, C and D are the Sichuan chinaberry extract and biomedical uses of active ingredient.
Background technology
Meliasenin B, C and D are isolated three kinds of root of Beijing euphorbia alkane type triterpenoid (bibliography from Fructus meliae toosendan：
Limonoids and Triterpenoids from the Stem Bark ofMelia toosendan,
J.Nat.Prod.2010,73,664-668)。
At present, the pharmacological activity and medical usage not on Meliasenin B, C and D are reported.But but there is research
Show to include but is not limited to compound (the reference text related to the hepatotoxicity wind agitation of Fructus meliae toosendan including Meliasenin B in Fructus meliae toosendan
Offer：In Fructus meliae toosendan hepatotoxicity wind agitation component rapid screening research, CHINA JOURNAL OF CHINESE MATERIA MEDICA, in June, 2013 o. 11th of volume 38).
Project team finds that Meliasenin B, C and D have the activity of anti-liver cancer and anti-in the compound of screening treatment liver cancer,
It specify that its molecular mechanism, have studied toxicity of these three compounds to normal liver cell, and provide method of attenuating and (applied
Patent).
But Meliasenin B, the manufacturing cost of C and D monomers are of a relatively high, if can prepare it is a kind of with
Meliasenin B, C and D are the extract of main component, then can reduce cost of being used as medicine.
The content of the invention
It is an object of the invention to provide it is a kind of using Meliasenin B, C and D as the Sichuan chinaberry extract of active ingredient and
Its preparation method and biomedical uses.
The above-mentioned purpose of the present invention is achieved by following technical solution：
A kind of Sichuan chinaberry extract using Meliasenin B, C and D as active ingredient, Meliasenin B, C and D
The sum of mass percentage be not less than 75%.
The preparation method of above-mentioned Sichuan chinaberry extract, includes the following steps：
Step S1, solvent extraction：Fructus meliae toosendan is crushed, the extraction of 50-95% ethanol, extracting solution filtering, be concentrated into no alcohol taste；
Step S2, resin concentration：Concentrate obtained by step S1 is splined on AB-8 macroporous absorbent resins, first with 30% ethanol
10-14 column volume is eluted, then with 65% ethanol elution, the 7-9 column volume eluent of collection, is concentrated to dryness；
Step S3, molecule are fished：The reagent of fishing of enriched product obtained by step S2 is extracted, 3-5 is used after extracting solution filtering
The dichloromethane extraction of times volume, stands, precipitation, collects supernatant, be concentrated to dryness up to the extract；
Wherein, the reagent of fishing is choline chloride and zinc chloride according to molar ratio 1:3 eutectic solvents formed.
Above-mentioned Sichuan chinaberry extract is used as the biomedical uses of Gli1 gene expression inhibitors.
Above-mentioned Sichuan chinaberry extract is used as the biomedical uses of Gli2 gene expression inhibitors.
Above-mentioned Sichuan chinaberry extract is used as the biomedical uses for preparing the medicine of prevention liver cancer.
A kind of pharmaceutical composition, including above-mentioned Sichuan chinaberry extract, further include naringenin.
Aforementioned pharmaceutical compositions are used as the biomedical uses of Gli1 gene expression inhibitors.
Aforementioned pharmaceutical compositions are used as the biomedical uses of Gli2 gene expression inhibitors.
Aforementioned pharmaceutical compositions are used as the biomedical uses for preparing the medicine of prevention liver cancer.
A kind of pharmaceutical preparation, including above-mentioned Sichuan chinaberry extract, or aforementioned pharmaceutical compositions, further including can pharmaceutically connect
The carrier or excipient received, are made pharmaceutically acceptable formulation.
Advantages of the present invention：
1st, Sichuan chinaberry extract provided by the invention can to significantly inhibit liver cancer thin by suppressing Gli1, Gli2 gene expression
The propagation of born of the same parents, and obvious dose dependent is presented in inhibitory action, which presses down for the effective of Gli1, Gli2 gene
Preparation, can be used for the medicine for preparing prevention liver cancer.One skilled in the art will appreciate that it is one to suppress Gli1, Gli2 gene expression
The more mature anti-cancer molecules path of kind, the Sichuan chinaberry extract are exactly based on this path and play antihepatocarcinoma effect.The present invention
It has also been found that naringenin can reduce toxicity of the Sichuan chinaberry extract of the present invention to Human normal hepatocyte, but it is thin to liver cancer not influence it
The toxicity of born of the same parents.
2nd, provided by the present invention for preparing the method for above-mentioned Sichuan chinaberry extract without using silica gel column chromatography, Mei Yibu
Industrialized production is suitable for, implementation is strong.
Brief description of the drawings
Fig. 1 is the HPLC chromatogram of extracting solution obtained by step S1 in embodiment 1；
Fig. 2 is the HPLC chromatogram of eluent obtained by step S2 in embodiment 1；
Fig. 3 is the HPLC chromatogram that step S3 is extract obtained in embodiment 1；
Fig. 4 is proliferation inhibition rate of the basic, normal, high dosage of Sichuan chinaberry extract to HepG2 cells；
Fig. 5 is the relative expression quantity of Gli1mRNA, Gli2mRNA in each administration group HepG2 cells；
Fig. 6 is Gli1 albumen, the relative expression quantity of Gli2 albumen in each administration group HepG2 cells；
Fig. 7 is proliferation inhibition rate of the basic, normal, high dosage of Sichuan chinaberry extract to SMMC-7721 cells；
Fig. 8 is the relative expression quantity of Gli1mRNA, Gli2mRNA in each administration group SMMC-7721 cells；
Fig. 9 is Gli1 albumen, the relative expression quantity of Gli2 albumen in each administration group SMMC-7721 cells.
Embodiment
The specific guarantor for introducing essentiality content of the present invention, but the present invention not being limited with this with reference to the accompanying drawings and examples
Protect scope.
Embodiment 1：The preparation and constituent analysis of Sichuan chinaberry extract
1st, the preparation of Sichuan chinaberry extract
Fructus meliae toosendan is purchased from Bozhou Chinese Medicinal Materials Markets, is identified as Meliaceae plant melia toosendan MeLia toosendan
The dry mature fruit of Sieb.et Zucc..It is spare to be crushed to 60 mesh.
The preparation of eutectic solvent：By choline chloride and zinc chloride according to molar ratio 1:3 mixing, 80 DEG C of heating stirrings to shape
Into transparent and homogeneous liquid, hermetically drying preserves, spare.The eutectic solvent is used as the reagent of fishing of Meliasenin B, C and D.
The preparation method of Sichuan chinaberry extract, includes the following steps：
Step S1, solvent extraction：By ethanol water that Fructus meliae toosendan and concentration expressed in percentage by volume are 70% according to solid-liquid ratio 1:
30 cold soakings extract 3 times, each 24h, merge extract, extracting solution filtering, be concentrated into no alcohol taste；
Step S2, resin concentration：Concentrate obtained by step S1 is splined on AB-8 macroporous absorbent resins, first uses volume basis
The ethanol water that concentration is 30% elutes 12 column volumes, then is eluted with the ethanol water that concentration expressed in percentage by volume is 65%,
The 7-9 column volume eluent is collected, is concentrated to dryness；
Step S3, molecule are fished：The reagent of fishing of enriched product obtained by step S2 is extracted, solid-liquid ratio 1:5, stirring carries
Take 2 it is small when, absorbent cotton filtering, when filtrate is small with the dichloromethane stirring extraction 0.5 of 4 times of volumes, stand, precipitation, filter or from
The heart, collects filtrate or centrifuged supernatant, is concentrated to dryness up to the extract.
Wherein, step S1 concentration of alcohol can be 50-95%, and the concentration of alcohol of step S2 elution enriched substances can be 60-
70%；Step S3 extracting solutions can be 1 with methylene chloride volume ratio:3-5.
2nd, HPLC is analyzed
Chromatographic condition：
Agilent 1100LC series liquid chromatograph instrument；Symmetry C18 chromatographic columns (4.6mm × 250mm, 5 μm)；
Mobile phase：A phases are the water containing 1% formic acid；B phases are the acetonitrile containing 1% formic acid；
Gradient elution program：0-10min, 25-40%B；10-20min, 40%B；20-35min, 40%-48%B；
Flow velocity l mL/min；Detection wavelength 226nm；35 DEG C of column temperature, 10 μ L of sampling volume.
The HPLC chromatogram of extracting solution is as shown in Figure 1 obtained by step S1；
The HPLC chromatogram of eluent is as shown in Figure 2 obtained by step S2；
HPLC chromatogram extract obtained step S3 is as shown in Figure 3.
It can be seen that from Fig. 1-3, the total content of Meliasenin B, C and D in the product that each step of the method for the present invention obtains
Higher and higher, the chromatogram of the extract finally obtained is as shown in figure 3, main component is Meliasenin B, C and D, through external standard
Standard measure understands that the sum of content of Meliasenin B, C and D is 76.9%.
Embodiment 2：The influence that Sichuan chinaberry extract breeds human hepatoma HepG2 cell
First, experiment material
Human hepatoma HepG2 cell's strain is purchased from Shanghai Ji Kai gene technology Co., Ltd；
DMEM culture mediums, hyclone and PBS are purchased from Gibco companies；TRIzol is purchased from Invitrogen companies；Reverse transcription
Kit is purchased from Fermentas companies of the U.S., and PCR kit for fluorescence quantitative is purchased from TaKaRa treasured bioengineering (Dalian) limited public affairs
Department；Cell pyrolysis liquid, SDS-PAGE albumen sample-loading buffer (5 ×), BCA protein concentrations test kit, 20 × TPBS bufferings
Liquid etc. is purchased from the green skies biotechnology research institute in Jiangsu, and pvdf membrane is purchased from Millipore companies of the U.S.；CCK-8 kits are purchased from
Japanese colleague's chemistry institute；Gli1, Gli2 and GAPDH primer are synthesized by Shanghai Sheng Gong bio-engineering corporations；
Rabbit-anti people Gli1 monoclonal antibodies, rabbit-anti people Gli2 monoclonal antibodies are purchased from U.S. Santa Cruz
Biotechnology, the anti-human GAPDH monoclonal antibodies of mouse are purchased from Beijing Bioisystech Co., Ltd of Zhong Shan Golden Bridge；
Sichuan chinaberry extract (EXTR) is prepared by embodiment 1.
2nd, experimental method
1st, human hepatoma HepG2 cell cultivates
HepG2 cell lines are in 37 DEG C, 5%CO2, under the conditions of saturated humidity, be placed in containing 10% hyclone,
Secondary culture in the DMEM culture mediums of 100U/mL penicillin and 100U/mL streptomysins, is passed on 1 time, growth period of taking the logarithm is thin per 2d
Born of the same parents.
2nd, experiment packet
EXTR low dosages administration group (5mg/L)；
EXTR middle dosages administration group (10mg/L)；
EXTR high doses administration group (20mg/L)；
Control group：HepG2 cells without any drug-treated.
3rd, CCK-8 methods measure cell proliferation inhibition rate
It is with reference to kit specification, the HepG2 of exponential phase is cells trypsinised, centrifugation, with containing 10%
The DMEM culture mediums of hyclone are made into cell suspension, and being seeded to 96 orifice plates makes cell density be 5 × 104/ mL is wet in saturation
Degree, 37 DEG C, 5%CO2Under conditions of culture 24h make cell attachment.The whole mass concentration dosing as needed for above-mentioned packet, control group is only
Add the PBS of pH7.4, each mass concentration sets 3 multiple holes, and CCK-8 48h after processing are added, per 10 μ L of hole, under the same terms
Continue to cultivate 2h, measure the absorbance in each hole under 450nm wavelength with microplate reader, inhibiting rate is calculated as follows.Inhibiting rate=
(1- administration groups absorbance/control group absorbance) × 100%.
4th, real-time fluorescence quantitative PCR detection Gli1mRNA, Gli2mRNA expression
Using 5.0 software Design primers of Primer, Gli1 upstream region of gene primers：5'-TCCAGCTAGAGTCCAGAGGT-
3', anti-sense primer：5'-TGGCTTGACTTGCACTTGTC-3'；Gli2 upstream region of gene primers：5'-
TGGCCGCTTCAGATGACAGATGTTG-3', anti-sense primer：5'-CGTTAGCCGAATGTCAGCCGTGAAG-3'；Internal reference
GAPDH sense primers：5'-AAGAAGGTGGTGAAGCAGGC-3', anti-sense primer：5'-ACCACCCTGTTGCTGTAGCC-3'.
Primer is synthesized by Shanghai Sheng Gong bio-engineering corporations.
Single cell suspension is made in HepG2 cells in exponential phase, is counted, is planted in 6 orifice plates, per hole cell number about
For 1 × 106It is a；After cell attachment, cultivate to 80% fusion, change serum free medium synchronization；Add afterwards in 6 orifice plates
Enter the Sichuan chinaberry extract of various concentrations, be put into incubator and cultivate 24h (control group not dosing)；Culture medium is discarded, is washed with PBS
Wash 2 times.TRIzol methods extract each group cell total rna respectively, and reverse transcription is cDNA.Set reaction condition be：95 DEG C of pre-degenerations
30s；95 DEG C of denaturation 5s, 60 DEG C of annealing 30s, totally 40 circulate.The target gene and reference gene of each sample are expanded at the same time.Often
Group 3 repeating holes of cell design.Using 2-△△CtAnalytic approach, is corrected by GAPDH gene levels.Ct values is in each reaction tubes
Fluorescence signal circulation for being undergone when reaching the thresholding of setting, it is average by formula △ CT=CT average values (target gene)-CT
It is worth the △ CT that (reference gene) calculates administration group and control group respectively, then is compareed by formula △ △ CT=△ CT administration group-△ CT
Group, calculates 2-△△Ct, 2-△△CtIt is the relative expression quantity of Gli1mRNA, Gli2mRNA in each administration group HepG2 cells.
5th, Westernblot experiments detection Gli1 albumen, Gli2 protein expression levels
Single cell suspension is made in HepG2 cells in exponential phase, is counted, is planted in 6 orifice plates, per hole cell number about
For 1 × 106It is a；After cell attachment, cultivate to 80% fusion, change serum free medium synchronization；Add afterwards in 6 orifice plates
Enter the Sichuan chinaberry extract of various concentrations, be put into incubator and cultivate 24h (control group not dosing)；Culture medium is discarded, is washed with PBS
Wash 2 times；Cell pyrolysis liquid is added, attached cell is gently scraped with cell scraper, is drawn in EP pipes, 4 DEG C, 15000rpm centrifugations
15min, draws supernatant and obtains total protein of cell, BCA methods carry out protein quantification；SDS is carried out per 20 μ g loadings of hole with total protein concentration
PAGE electrophoresis, transferring film；5% skimmed milk power is prepared with TPBS, pvdf membrane is closed, is positioned over 2h in 37 DEG C of shaking tables；By pvdf membrane plus
Enter by 1:In 1000 diluted GAPDH, Gli1, Gli2 antibody, 4 DEG C of shaking tables are incubated overnight；Add by after proper proportion dilution
Secondary antibody, 37 DEG C of incubation 2h；ECL developer solutions are spread evenly across band relevant position on film, in chemiluminescence imaging analyzer
Exposure；Interpretation of result is carried out using Image J image analysis softwares, measures the gray value of band, calculates the purpose band of each group
With the ratio of internal reference, compare Gli1 albumen, Gli2 protein diversities between each group.
6th, statistical analysis
Statistical disposition is carried out to the data obtained using 19.0 softwares of SPSS, data are represented with mean value ± deviation, measurement data
Group difference compare using One-WayANOVA.
3rd, experimental result
1st, influence of the Sichuan chinaberry extract to HepG2 cell Proliferations
Compared with control group, the HepG2 cell Proliferations of the basic, normal, high dosage administration group of Sichuan chinaberry extract are substantially suppressed,
Absorbance significantly reduces (P ＜ 0.05).Table 1 and Fig. 4 are propagation of the basic, normal, high dosage of Sichuan chinaberry extract to HepG2 cells
Inhibiting rate, it can be seen that obvious concentration dependent is presented in inhibitory action.
Proliferation inhibition rate of the basic, normal, high dosage of 1 Sichuan chinaberry extract of table to HepG2 cells
2nd, influence of the Sichuan chinaberry extract to Gli1mRNA, Gli2mRNA expression in HepG2 cells
Compared with control group, Gli1mRNA in the HepG2 cells of the basic, normal, high dosage administration group of Sichuan chinaberry extract,
Gli2mRNA expression is obvious to be suppressed, and target gene significantly reduces (P ＜ 0.05 or P ＜ with respect to the expression quantity of reference gene
0.01).Table 2 and Fig. 5 are Gli1mRNA, Gli2mRNA in the basic, normal, high dosage administration group HepG2 cells of Sichuan chinaberry extract
Relative expression quantity, it can be seen that obvious concentration dependent is presented in inhibitory action.
The relative expression quantity of Gli1mRNA, Gli2mRNA in each administration group HepG2 cells of table 2
3rd, influence of the Sichuan chinaberry extract to Gli1 albumen, Gli2 protein expression levels in HepG2 cells
Compared with control group, Gli1 albumen, Gli2 in the HepG2 cells of the basic, normal, high dosage administration group of Sichuan chinaberry extract
Protein expression is substantially suppressed.Table 3 and Fig. 6 are Gli1 eggs in the basic, normal, high dosage administration group HepG2 cells of Sichuan chinaberry extract
In vain, the relative expression levels of Gli2 albumen, it can be seen that obvious concentration dependent is presented in inhibitory action.
Gli1 albumen, the relative expression quantity of Gli2 albumen in each administration group HepG2 cells of table 3
Above-described embodiment test result indicates that, Sichuan chinaberry extract can be shown by suppressing Gli1, Gli2 gene expression
The propagation for suppressing HepG2 cells is write, and obvious dose dependent, Sichuan chinaberry extract Gli1, Gli2 is presented in inhibitory action
Effective inhibitor of gene, can be used for the medicine for preparing prevention liver cancer.
Embodiment 3：Influence of the Sichuan chinaberry extract to human liver cancer SMMC-7721 systems cell Proliferation
First, experiment material
SMMC-7721 cell line is purchased from Shanghai Ji Kai gene technology Co., Ltd；
DMEM culture mediums, hyclone and PBS are purchased from Gibco companies；TRIzol is purchased from Invitrogen companies；Reverse transcription
Kit is purchased from Fermentas companies of the U.S., and PCR kit for fluorescence quantitative is purchased from TaKaRa treasured bioengineering (Dalian) limited public affairs
Department；Cell pyrolysis liquid, SDS-PAGE albumen sample-loading buffer (5 ×), BCA protein concentrations test kit, 20 × TPBS bufferings
Liquid etc. is purchased from the green skies biotechnology research institute in Jiangsu, and pvdf membrane is purchased from Millipore companies of the U.S.；CCK-8 kits are purchased from
Japanese colleague's chemistry institute；Gli1, Gli2 and GAPDH primer are synthesized by Shanghai Sheng Gong bio-engineering corporations；
Rabbit-anti people Gli1 monoclonal antibodies, rabbit-anti people Gli2 monoclonal antibodies are purchased from U.S. Santa Cruz
Biotechnology, the anti-human GAPDH monoclonal antibodies of mouse are purchased from Beijing Bioisystech Co., Ltd of Zhong Shan Golden Bridge；
Sichuan chinaberry extract (EXTR) is prepared by embodiment 1.
2nd, experimental method
1st, human hepatocarcinoma BEL-7402 culture
Human hepatoma cell strain SMMC-7721 is in 37 DEG C, 5%CO2Under the conditions of, it is placed in blue or green containing 10% hyclone, 100U/mL
Secondary culture in the DMEM culture mediums of mycin and 100U/mL streptomysins, is passed on 1 time, growth period cell of taking the logarithm per 2d.
2nd, experiment packet
EXTR low dosages administration group (5mg/L)；
EXTR middle dosages administration group (10mg/L)；
EXTR high doses administration group (20mg/L)；
Control group：SMMC-7721 cells without any drug-treated.
3rd, CCK-8 methods measure cell proliferation inhibition rate
It is with reference to kit specification, the SMMC-7721 of exponential phase is cells trypsinised, centrifugation, with containing
The DMEM culture mediums of 10% hyclone are made into cell suspension, and being seeded to 96 orifice plates makes cell density be 5 × 104/ mL, in saturation
Humidity, 37 DEG C, 5%CO2Under conditions of culture 24h make cell attachment.Whole mass concentration dosing, control group as needed for above-mentioned packet
Only plus pH7.4 PBS, each mass concentration sets 3 multiple holes, and CCK-8 48h after processing are added, per 10 μ L of hole, the same terms
Under continue cultivate 2h, the absorbance in each hole is measured under 450nm wavelength with microplate reader, inhibiting rate is calculated as follows.Inhibiting rate
=(1- administration groups absorbance/control group absorbance) × 100%.
4th, real-time fluorescence quantitative PCR detection Gli1mRNA, Gli2mRNA expression
Using 5.0 software Design primers of Primer, Gli1 upstream region of gene primers：5'-TCCAGCTAGAGTCCAGAGGT-
3', anti-sense primer：5'-TGGCTTGACTTGCACTTGTC-3'；Gli2 upstream region of gene primers：5'-
TGGCCGCTTCAGATGACAGATGTTG-3', anti-sense primer：5'-CGTTAGCCGAATGTCAGCCGTGAAG-3'；Internal reference
GAPDH sense primers：5'-AAGAAGGTGGTGAAGCAGGC-3', anti-sense primer：5'-ACCACCCTGTTGCTGTAGCC-3'.
Primer is synthesized by Shanghai Sheng Gong bio-engineering corporations.
Single cell suspension is made in SMMC-7721 cells in exponential phase, is counted, is planted in 6 orifice plates, per hole cell
Number about 1 × 106It is a；After cell attachment, cultivate to 80% fusion, change serum free medium synchronization；Afterwards in 6 orifice plates
The middle Sichuan chinaberry extract for adding various concentrations, is put into incubator and cultivates 24h (control group not dosing)；Culture medium is discarded, is used
PBS is washed 2 times.TRIzol methods extract each group cell total rna respectively, and reverse transcription is cDNA.Set reaction condition be：95 DEG C pre-
It is denatured 30s；95 DEG C of denaturation 5s, 60 DEG C of annealing 30s, totally 40 circulate.The target gene and internal reference base of each sample are expanded at the same time
Cause.3 repeating holes of every group of cell design.Using 2-△CtAnalytic approach, is corrected by GAPDH gene levels.Ct values are each reaction
Fluorescence signal in pipe reaches the circulation undergone during the thresholding of setting and is put down by formula △ CT=CT average values (target gene)-CT
Average (reference gene) calculates the △ CT of administration group and control group respectively, and as each group Gli1mRNA, Gli2mRNA is with respect to internal reference
The relative expression quantity of gene.
5th, Western blot experiments detection Gli1 albumen, Gli2 protein expression levels
Single cell suspension is made in SMMC-7721 cells in exponential phase, is counted, is planted in 6 orifice plates, per hole cell
Number about 1 × 106It is a；After cell attachment, cultivate to 80% fusion, change serum free medium synchronization；Afterwards in 6 orifice plates
The middle Sichuan chinaberry extract for adding various concentrations, is put into incubator and cultivates 24h (control group not dosing)；Culture medium is discarded, is used
PBS is washed 2 times；Add cell pyrolysis liquid, gently scrape attached cell with cell scraper, be drawn in EP pipes, 4 DEG C, 15000rpm from
Heart 15min, draws supernatant and obtains total protein of cell, BCA methods carry out protein quantification；Carried out with total protein concentration per 20 μ g loadings of hole
SDS PAGE electrophoresis, transferring film；5% skimmed milk power is prepared with TPBS, pvdf membrane is closed, is positioned over 2h in 37 DEG C of shaking tables；By PVDF
Film, which adds, presses 1:In 1000 diluted GAPDH, Gli1, Gli2 antibody, 4 DEG C of shaking tables are incubated overnight；Add and diluted by proper proportion
Secondary antibody afterwards, 37 DEG C of incubation 2h；ECL developer solutions are spread evenly across band relevant position on film, are analyzed in chemiluminescence imaging
Exposed in instrument；Interpretation of result is carried out using Image J image analysis softwares, measures the gray value of band, calculates the purpose of each group
The ratio of band and internal reference, compares Gli1 albumen, Gli2 protein diversities between each group.
6th, statistical analysis
Statistical disposition is carried out to the data obtained using 19.0 softwares of SPSS, data are represented with mean value ± deviation, measurement data
Group difference compare using One-WayANOVA.
3rd, experimental result
1st, influence of the Sichuan chinaberry extract to SMMC-7721 cell Proliferations
Compared with control group, the obvious quilt of the SMMC-7721 cell Proliferations of the basic, normal, high dosage administration group of Sichuan chinaberry extract
Suppress, absorbance significantly reduces (P ＜ 0.05).Table 4 and Fig. 7 are the basic, normal, high dosage of Sichuan chinaberry extract to SMMC-7721
The proliferation inhibition rate of cell, it can be seen that obvious concentration dependent is presented in inhibitory action.
Proliferation inhibition rate of the basic, normal, high dosage of 4 Sichuan chinaberry extract of table to SMMC-7721 cells
2nd, Sichuan chinaberry extract influences Gli1mRNA, Gli2mRNA expression in SMMC-7721 cells
Compared with control group, Gli1mRNA in the SMMC-7721 cells of the basic, normal, high dosage administration group of Sichuan chinaberry extract,
Gli2mRNA expression is obvious to be suppressed, and target gene significantly reduces (P ＜ 0.05 or P ＜ with respect to the expression quantity of reference gene
0.01).Table 5 and Fig. 8 be Gli1mRNA in the basic, normal, high dosage administration group SMMC-7721 cells of Sichuan chinaberry extract,
The relative expression quantity of Gli2mRNA, it can be seen that obvious concentration dependent is presented in inhibitory action.
The relative expression quantity of Gli1mRNA, Gli2mRNA in each administration group SMMC-7721 cells of table 5
3rd, influence of the Sichuan chinaberry extract to Gli1 albumen, Gli2 protein expression levels in SMMC-7721 cells
Compared with control group, Gli1 albumen in the SMMC-7721 cells of the basic, normal, high dosage administration group of Sichuan chinaberry extract,
Gli2 protein expressions are substantially suppressed.Table 6 and Fig. 9 are the basic, normal, high dosage administration group SMMC-7721 cells of Sichuan chinaberry extract
Middle Gli1 albumen, the relative expression levels of Gli2 albumen.
Gli1 albumen, the relative expression quantity of Gli2 albumen in each administration group SMMC-7721 cells of table 6
Above-described embodiment test result indicates that, Sichuan chinaberry extract can be shown by suppressing Gli1, Gli2 gene expression
The propagation of work suppression SMMC-7721 cells, and the obvious dose dependent of inhibitory action presentation, Sichuan chinaberry extract Gli1,
Effective inhibitor of Gli2 genes, can be used for the medicine for preparing prevention liver cancer.
Embodiment 3：Influence of the Sichuan chinaberry extract to hepatocellular carcinoma in nude mice growth of transplanted human
By the good exponential phase HepG2 cells of growth conditions, after pancreatin digestion, centrifugation and trypan blue count, physiology is used
Brine is made 1 × 107The cell suspension of a/mL, is inoculated at the right oxter 0.5cm of nude mice, every nude inoculation 0.1mL is thin respectively
Born of the same parents' suspension.Transplanted tumor in nude mice vernier caliper measurement transplantable tumor diameter, treats tumour growth to about 100mm3When nude mice is divided at random
For Sichuan chinaberry extract group and control group, every group 10.Sichuan chinaberry extract group dosage regimen is：Gastric infusion, 35mg/kg/
D, 1 time a day, control group give isometric solvent 0.5%CMC-Na.Administration three weeks.After last time administration 6h, nude mice is put to death,
Tumor mass is stripped to weigh.According to administration group and the equal re-computation administration group tumor control rate (such as table 7) of control group tumor mass.
7 Sichuan chinaberry extract group tumor control rate of table
| Sichuan chinaberry extract group | |
| In-vivo tumour inhibiting rate (%) | 62.8 |
Above-described embodiment test result indicates that, Sichuan chinaberry extract has the function that to suppress liver cancer in vivo.
Embodiment 4：Toxicity and naringenin attenuation of the Sichuan chinaberry extract to normal liver cell
Human normal liver cell L 02 cell line is purchased from China typical culture collection center, is containing 10% hyclone
In RPMI1640 culture mediums, 37 DEG C, 5%CO2Cellar culture is carried out, experiment is carried out in exponential phase cells.
It is with reference to kit specification, the L02 of exponential phase is cells trypsinised, centrifugation, with containing 10% tire
1640 culture mediums of RPMI of cow's serum are made into cell suspension, and being seeded to 96 orifice plates makes cell density be 3.5 × 104/ mL, full
With humidity, 37 DEG C, 5%CO2Under conditions of culture 24h make cell attachment.Whole mass concentration dosing, control as needed for following packet
Group only adds the PBS of pH7.4, and each mass concentration sets 3 multiple holes, and CCK-8 48h after processing are added, per 10 μ L of hole, identical bar
Continue to cultivate 2h under part, measure the absorbance in each hole under 450nm wavelength with microplate reader, calculate inhibiting rate.
Sichuan chinaberry extract group：20mg/L Sichuan chinaberry extracts；
Sichuan chinaberry extract attenuation group：+ 5 μM of naringenins of 20mg/L Sichuan chinaberry extracts.
Each group medicine is as shown in table 8 to the inhibiting rate of human normal liver cell L 02 cell.
Inhibiting rate of the 8 each group medicine of table to human normal liver cell L 02 cell
| Group | Inhibiting rate (%) | Group | Inhibiting rate (%) |
| Sichuan chinaberry extract group | 52.8±3.3 | Sichuan chinaberry extract attenuation group | 11.5±2.9 |
Above-described embodiment test result indicates that, Sichuan chinaberry extract also has obvious toxicity to Human normal hepatocyte,
Naringenin can significantly reduce toxicity of the Sichuan chinaberry extract to Human normal hepatocyte.One skilled in the art will appreciate that in tumour
Therapy field, toxicity i.e. its activity of many antitumor drugs, therefore, project team is tested using the method in embodiment 2 and 3
Whether the composition of Sichuan chinaberry extract and naringenin still has inhibited proliferation to human liver cancer cell, as a result with HepG2
Exemplified by, inhibiting rate is respectively (52.1 ± 2.7) % after+5 μM of naringenins of 20mg/L Sichuan chinaberry extracts act on HepG248h, with
The inhibiting rate of naringenin is not added with without significant difference (P ＞ 0.05).This explanation naringenin can reduce Sichuan chinaberry extract to people just
The toxicity of normal liver cell, but Sichuan chinaberry extract is not influenced to liver cancer cells toxicity.
Embodiment 6：Prevent the pharmaceutical preparation of liver cancer
1st, Sichuan chinaberry extract tablet
Sichuan chinaberry extract 90g, starch 180g, magnesium stearate 3g.
Preparation process：Sichuan chinaberry extract adds starch, magnesium stearate to be uniformly mixed, and particle is made, dry, tabletting.
2nd, Sichuan chinaberry extract capsule
Sichuan chinaberry extract 90g, starch 180g, magnesium stearate 3g.
Preparation process：Sichuan chinaberry extract adds starch, magnesium stearate to be uniformly mixed, and particle is made, dry, encapsulated.
3rd, Sichuan chinaberry extract and naringenin composite tablet
Sichuan chinaberry extract 90g, naringenin 10g, starch 180g, magnesium stearate 3g.
Preparation process：Sichuan chinaberry extract and naringenin add starch, magnesium stearate to be uniformly mixed granulation, dry, tabletting.
4th, Sichuan chinaberry extract and naringenin compound capsule
Sichuan chinaberry extract 90g, naringenin 10g, starch 180g, magnesium stearate 3g.
Preparation process：Sichuan chinaberry extract and naringenin add starch, magnesium stearate to be uniformly mixed granulation, and drying is encapsulated.
The effect of above-described embodiment is the essentiality content for specifically introducing the present invention, but those skilled in the art should know
Protection scope of the present invention, should not be confined to the specific embodiment by road.
Claims (10)
- A kind of 1. Sichuan chinaberry extract using Meliasenin B, C and D as active ingredient, it is characterised in that：It is described The sum of mass percentage of Meliasenin B, C and D is not less than 75%.
- 2. the preparation method of Sichuan chinaberry extract described in claim 1, it is characterised in that include the following steps：Step S1, solvent extraction：Fructus meliae toosendan is crushed, the extraction of 50-95% ethanol, extracting solution filtering, be concentrated into no alcohol taste；Step S2, resin concentration：Concentrate obtained by step S1 is splined on AB-8 macroporous absorbent resins, first with 30% ethanol elution 10-14 column volume, then with 65% ethanol elution, collect the 7-9 column volume eluent, be concentrated to dryness；Step S3, molecule are fished：The reagent of fishing of enriched product obtained by step S2 is extracted, with 3-5 times of body after extracting solution filtering Long-pending dichloromethane extraction, stands, precipitation, collects supernatant, be concentrated to dryness up to the extract；Wherein, the reagent of fishing is choline chloride and zinc chloride according to molar ratio 1:3 eutectic solvents formed.
- 3. the Sichuan chinaberry extract described in claim 1 is used as the biomedical uses of Gli1 gene expression inhibitors.
- 4. the Sichuan chinaberry extract described in claim 1 is used as the biomedical uses of Gli2 gene expression inhibitors.
- 5. the Sichuan chinaberry extract described in claim 1 is used as the biomedical uses for preparing the medicine of prevention liver cancer.
- A kind of 6. pharmaceutical composition, it is characterised in that：Including the Sichuan chinaberry extract described in claim 1, naringenin is further included.
- 7. the pharmaceutical composition described in claim 6 is used as the biomedical uses of Gli1 gene expression inhibitors.
- 8. the pharmaceutical composition described in claim 6 is used as the biomedical uses of Gli2 gene expression inhibitors.
- 9. the pharmaceutical composition described in claim 6 is used as the biomedical uses for preparing the medicine of prevention liver cancer.
- A kind of 10. pharmaceutical preparation, it is characterised in that：Including the Sichuan chinaberry extract described in claim 1, or claim 6 institute The pharmaceutical composition stated, further includes pharmaceutically acceptable carrier or excipient, pharmaceutically acceptable formulation is made.
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| BE1027352B1 (en) * | 2019-06-25 | 2021-01-20 | Univ Fuyang Normal | Process for extracting total flavone from the seeds of sweet cedar |
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