CN105106870A - Medicinal composition for treating colorectal cancer and preparation method and application thereof - Google Patents
Medicinal composition for treating colorectal cancer and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a medicinal composition for treating colorectal cancer. The medicinal preparation is prepared by adopting the following medicinal materials in part by weight: 18 to 22 parts of turtle shell, 18 to 22 parts of oldenlandia diffusa, 16 to 20 parts of curcuma zedoary, 16 to 20 parts of astragalus membranaceus. The invention also provides a preparation method of the medicinal composition. The medicinal effect experiment proves that the medicinal composition has a remarkable effect for inhibiting the multiplication of colorectal cancer epithelial cells HCT116, and a novel choice is provided for the clinic.
Description
Technical field
The present invention relates to a kind of pharmaceutical composition for the treatment of colorectal cancer and preparation method thereof, purposes, belong to field of medicaments.
Background technology
Cancer is commonly encountered diseases and the frequently-occurring disease of a kind of serious threat human health and life, and the dead shelter that the mankind cause because of cancer has the second of mortality, is only second to cardiovascular and cerebrovascular disease.The pathogenesis of cancer has factors to participate in, think at present apoptosis suppressed be one of them approach.Apoptosis is a kind of programmed cell death, shows as that cellular morphology reduces, chromatin agglutination, nuclear collapse, chromatin dna be hydrolyzed into fragment (180bp multiple), form apoptotic body.Apoptosis is early stage, and mitochondrial membrane potential in anoxic reduces, and cytochrome C is transferred to Cell sap in mitochondrion subsequently, and combines with APAF1 (apoptosis protease-activating factor 1), and isochrone mitochondria function fails.Subsequently, cytochrome C/APAF1 complex activates Caspase-9, the latter reactivation Caspase-3 and other downstream Caspase (caspase), causes Phosphatidylserine inside cell membrane (PS) to turn up.At this moment cell diminishes, chromatin pyknosis (chromatincondensation), DNA fragmentation (DNAfragmentation), forms apoptotic body, and cell bubbles (cellbubbling), apoptosis.When apoptosis deficiency, in cell colony, survival is destroyed with dead balance, and the length that has a net increase of of cancer cell count strengthens.
Chinese medicine has very consequence in cancer treatment procedure, and it can improve Cancer Patients ' Immune Function and anti-cancer ability, can also alleviate toxic and side effects during patient's Radiotherapy chemotherapy, improves patient to the toleration of chemicotherapy.Modern study shows, several kinds of Chinese medicinal materials and extract thereof all have the effect of prevention or Therapeutic cancer.Such as, curcumin can the proliferation and spreading of Tumor suppression; Drug for invigorating blood circulation and eliminating stasis Rhizoma Chuanxiong can suppress the transfer of murine melanoma; The plug injection liquid that the decocts such as Chinese medicine Radix Aconiti, Rhizoma Polygoni Cuspidati are made has obvious anticancer effect.Due to Chinese medicine complicated mechanism in Therapeutic cancer process, the effect that different crude drug compatibilities produces may not be identical.
Summary of the invention
The object of the present invention is to provide pharmaceutical composition of a kind for the treatment of colorectal cancer newly and preparation method thereof, purposes.
The invention provides a kind of pharmaceutical composition for the treatment of colorectal cancer, it is the preparation be prepared from by the crude drug of following weight proportioning: Carapax Trionycis 18 parts ~ 22 parts, Herba Hedyotidis Diffusae 18 parts ~ 22 parts, Rhizoma Curcumae 16 parts ~ 20 parts, the Radix Astragali 16 parts ~ 20 parts.
Preferably, described pharmaceutical composition is the preparation be prepared from by the crude drug of following weight proportioning: Carapax Trionycis 20 parts, Herba Hedyotidis Diffusae 20 parts, Rhizoma Curcumae 18 parts, the Radix Astragali 18 parts.
Further, described pharmaceutical composition is active component by the protogenic medicinal powder of Carapax Trionycis, Herba Hedyotidis Diffusae, Rhizoma Curcumae, the Radix Astragali, water or extractive with organic solvent, adds pharmaceutically acceptable adjuvant or complementary composition is prepared into preparation.
Further, described preparation is decoction, powder, pill, granule, tablet or oral liquid.
The invention provides a kind of method preparing described pharmaceutical composition, it comprises following steps:
A, get crude drug in proportion, smash;
B, use ethanol extraction, be prepared into extractum;
C, add pharmaceutically conventional adjuvant or complementary composition, be prepared into preparation.
Preferably, described ethanol is the ethanol water of 85%.
The invention provides described pharmaceutical composition and prepare the purposes treated and/or prevented in the medicine of colorectal cancer.
Further, described medicine is the medicine treating and/or preventing one or both cancers in rectal cancer, colon cancer.
Further, described medicine is the medicine suppressing colon cancer cell HCT116 propagation.
Further, described medicine is the activity improving in colon cancer cell HCT116 apoptosis enzyme Caspase-9, Caspase-3 one or both; Reduce the BCL-2 protein expression level of colon cancer cell HCT116; Improve the BAX protein expression level of colon cancer cell HCT116; There is apoptotic medicine in induction colon cancer cell HCT116.
Medicine of the present invention is the treatment specific to Carapax Trionycis, Herba Hedyotidis Diffusae, Rhizoma Curcumae and Radix Astragali proportioning being used for colorectal cancer, has effect of hard masses softening and resolving, heat-clearing and toxic substances removing, removing blood stasis Xiao Disorder, QI invigorating righting.Experiment proves, this pharmaceutical composition can significantly suppress Human colorectal carcinoma epithelial cell HCT116 to breed, and especially 500 μ g/ml dosage are suitable with 100 μMs of oleanolic acid action intensities, and can regulate immunity of organism.Drug therapy cancer of the present invention, drug effect is clear and definite, and cost is low, and crude drug source is in animals and plants, Clinical practice safety, avoids the side effect of radiotherapy and chemotherapy medicine, provides a kind of new medication selection for clinical.
Obviously, according to foregoing of the present invention, according to ordinary technical knowledge and the customary means of this area, not departing under the present invention's above-mentioned basic fundamental thought prerequisite, the amendment of other various ways, replacement or change can also be made.
The detailed description of the invention of form by the following examples, is described in further detail foregoing of the present invention again.But this should be interpreted as that the scope of the above-mentioned theme of the present invention is only limitted to following example.All technology realized based on foregoing of the present invention all belong to scope of the present invention.
Accompanying drawing explanation
Fig. 1 pharmaceutical composition of the present invention is on the impact of HCT116 cellular morphology
Detailed description of the invention
The preparation of embodiment 1 pharmaceutical composition of the present invention
A, weighting raw materials: Carapax Trionycis 20g, Herba Hedyotidis Diffusae 20g, Rhizoma Curcumae 18g, Radix Astragali 18g;
B, above-mentioned decoction pieces to be smashed, by 85% ethanol heating extraction twice, each 1h, merge twice medicinal liquid, be concentrated into finite concentration with Rotary Evaporators, volatilize solvent with evaporating dish, dry to constant weight in baking oven, the extraction yield of extractum is 20.43%, extractum: primary crude drug quality=10:2.043.
The preparation of embodiment 2 pharmaceutical composition of the present invention
A, weighting raw materials: Carapax Trionycis 18g, Herba Hedyotidis Diffusae 18g, Rhizoma Curcumae 16g, Radix Astragali 16g;
B, above-mentioned decoction pieces to be smashed, by 85% ethanol heating extraction twice, each 1h, merge twice medicinal liquid, be concentrated into finite concentration with Rotary Evaporators, volatilize solvent with evaporating dish, dry to constant weight in baking oven, the extraction yield of extractum is 20.43%, extractum: primary crude drug quality=10:1.968.
The preparation of embodiment 3 pharmaceutical composition of the present invention
A, weighting raw materials: Carapax Trionycis 22g, Herba Hedyotidis Diffusae 22g, Rhizoma Curcumae 20g, Radix Astragali 20g;
B, above-mentioned decoction pieces to be smashed, by 85% ethanol heating extraction twice, each 1h, merge twice medicinal liquid, be concentrated into finite concentration with Rotary Evaporators, volatilize solvent with evaporating dish, dry to constant weight in baking oven, the extraction yield of extractum is 20.43%, extractum: primary crude drug quality=10:2.129.
Get above-mentioned extractum and add pharmaceutically conventional adjuvant or complementary composition, be prepared into decoction, powder, pill, granule, tablet or oral liquid.
Beneficial effect of the present invention is further illustrated below by way of experimental example.Trionyx sinensis Wiegmann Serpentis Fighting cancer club alleged is below the compositions be prepared from according to embodiment 1.
[material]
Cell strain: human colon carcinoma epithelial cell HCT116, purchased from Chinese Academy of Sciences's Shanghai cell bank, frozen in liquid nitrogen.
Medicine: Carapax Trionycis, Herba Hedyotidis Diffusae, Rhizoma Curcumae, Radix Astragali decoction pieces purchased from company of Tongrentang, through being accredited as certified products.Key instrument: constant temperature CO
2cell culture incubator (Thermo); Multi-functional microplate reader (ThermoScientificVarioskanFlash, Type3001); AllegraX-12 type centrifuge (BeckmanCoulter); DMI3000B type fluorescence microscope (Leica); Superclean bench (Su Jing is safe and sound); The consumptive material such as Tissue Culture Flask, Tissue Culture Plate is provided by Corning.
Reagent: McCoy ' s5A culture medium (Sigma-Aldrich); Calf serum (Invitrogen); Trypsin Sigma-Aldrich); MTT test kit (the green skies); Caspase-9 and Caspase-3 detection kit (the green skies); Bradford determination of protein concentration test kit (the green skies); HumanBax (BCL-2-AssociatedXProtein) ELISA kit and HumanBCL-2 (B-celllymphoma2) ELISA kit (Elabscience).Hoechst33342 dyeing liquor (triumphant base is biological), fluorescence quenching (triumphant base is biological), other reagent is domestic analytical pure.
[method]
Cell culture
HCT116 cell is with containing McCoy ' the s5A complete medium of 10% calf serum at 5%CO
2, cultivate under 37 DEG C of wet heat conditions, and by 0.25% trypsin-EDTA had digestive transfer culture.
MTT cell proliferation experiment
HCT116 cell is inoculated in 96 well culture plates, every hole about 3 × 10
3individual cell.After inoculation 24h, with culture fluid process cell 12h, 24h, 36h, 48h containing variable concentrations Trionyx sinensis Wiegmann Serpentis Fighting cancer club (1000 μ g/ml, 500 μ g/ml, 250 μ g/ml, 125 μ g/ml, 62.5 μ g/ml, 37.25 μ g/ml, 0 μ g/ml), positive drug oleanolic acid (100 μMs), control solvent (0.4%DMSO), often organize 8 holes.Inhale subsequently and abandon culture fluid, every hole adds the 5mg/mlMTT of 100 μ l, hatches 4h, then adds the Formazan lysate of 100 μ l.Absorbance (A is surveyed by microplate reader at 570nm after 4 hours
570nm).Cell inhibitory rate (%)=(A
matched group-A
medicine group)/A
matched group× 100%.
The mensuration of apoptosis enzyme Caspase-9 and Caspase-3 activity
HCT116 cell is inoculated in 6 orifice plates, every hole about 1 × 10
5individual cell, with the culture fluid process cell 48h containing Trionyx sinensis Wiegmann Serpentis Fighting cancer club (125 μ g/ml, 250 μ g/ml, 500 μ g/ml), oleanolic acid (100 μMs) and control solvent (0.4%DMSO), digestion collecting cell, add protein lysate, and measure protein concentration (being undertaken by test kit description) by Bradford method.Caspase-3 and Caspase-9 Activity determination: add 50 μ l and detect buffer in 96 orifice plates, be mixed into 40 μ l testing samples again, finally add 10 μ lAc-LEHD-pNA (Caspase-9 mensuration) or 10 μ lAc-DEVD-pNA (Caspase-3 mensuration), mixing, 37 DEG C of overnight incubation.A is measured by microplate reader
405nm, and calculate enzyme activity.
The mensuration of BCL-2 and BAX gene expression dose
HCT116 cell is inoculated in 6 orifice plates, every hole about 1 × 10
5cell, with the culture fluid process cell 48h containing Trionyx sinensis Wiegmann Serpentis Fighting cancer club (125 μ g/ml, 250 μ g/ml, 500 μ g/ml), oleanolic acid (100 μMs) and control solvent (0.4%DMSO), draw culture fluid, centrifugal, get cell conditioned medium liquid, detect BCL-2 and BAX gene expression dose in cell culture fluid by BAX and BCL-2 test kit description.
Morphological observation
HCT116 cell is inoculated in 96 well culture plates, every hole about 5 × 10
3individual cell, with the culture fluid process cell 48h containing Trionyx sinensis Wiegmann Serpentis Fighting cancer club (125 μ g/ml, 250 μ g/ml, 500 μ g/ml), oleanolic acid (100 μMs) and control solvent (0.4%DMSO).Methanol fixed cell, PBS cleaning twice, with Hoechst33342 staining cell 5 minutes, after fluorescence quenching mounting, observation of cell form under fluorescence microscope.Statistical analysis: analyze by SPSS19.0 software statistics, all data acquisitions are used
represent, carry out one factor analysis of variance and t inspection.
[result]
The impact that experimental example 1 Trionyx sinensis Wiegmann Serpentis Fighting cancer club is bred Human colorectal carcinoma epithelial cell HCT116
The Trionyx sinensis Wiegmann Serpentis Fighting cancer club of table 1 various dose on the impact of HCT116 cell proliferation (
n=8)
(note: compare * P<0.05 with blank, * * P<0.01; #P<0.05 is compared, ##P<0.01 with oleanolic acid.)
Experiment shows, except 37.25 μ g/ml, other each dosage Trionyx sinensis Wiegmann Serpentis Fighting cancer club and positive drug oleanolic acid have significant inhibitory action to Human colorectal carcinoma epithelial cell HCT116 propagation, and the effect of Trionyx sinensis Wiegmann Serpentis Fighting cancer club shows certain dose dependent.Wherein, the Trionyx sinensis Wiegmann Serpentis Fighting cancer club of 500 μ g/ml and 1000 μ g/ml is suitable with 100 μMs of oleanolic acid action intensities, in table 1.So in following experiment, we choose 125 μ g/ml, 250 μ g/ml, 500 μ g/ml Trionyx sinensis Wiegmann Serpentis Fighting cancer club carry out the apoptotic research of induction HCT116.
Table 2 Trionyx sinensis Wiegmann Serpentis Fighting cancer club different action time on the impact of HCT116 cell proliferation (
n=8)
(note: compare * P<0.05 with 0h; #P<0.05 is compared with 12h; $ P<0.05 is compared with 24h; ▲ P<0.05 is compared with 36h.)
Experiment shows, 500 μ g/ml Trionyx sinensis Wiegmann Serpentis Fighting cancer club and 100 μMs of oleanolic acid of different action time all have significant inhibitory action to HCT116 cell proliferation, and present regular hour dependency, wherein the effect that shows of Trionyx sinensis Wiegmann Serpentis Fighting cancer club and oleanolic acid process cell 48h is the strongest, in table 2.So in following experiment, we set drug exposure times is 48h, research Trionyx sinensis Wiegmann Serpentis Fighting cancer club is on the apoptotic impact of HCT116.
The apoptosis enzyme Caspase-9 of experimental example 2 Trionyx sinensis Wiegmann Serpentis Fighting cancer club on HCT116 cell and the impact of Caspase-3 activity
Table 3 Trionyx sinensis Wiegmann Serpentis Fighting cancer club to HCT116 apoptosis enzyme Caspase-9 activity influence (
n=6)
(note: compare * P<0.05 with blank, * * P<0.01; #P<0.05 is compared, ##P<0.01 with oleanolic acid.)
Experiment shows, 125 μ g/ml, 250 μ g/ml, 500 μ g/ml Trionyx sinensis Wiegmann Serpentis Fighting cancer club and 100 μMs of oleanolic acid process HCT116 cell 48h, can significantly improve the activity of HCT116 apoptosis enzyme Caspase-9, and the effect of Trionyx sinensis Wiegmann Serpentis Fighting cancer club shows certain dose dependent.Wherein, the action intensity of 500 μ g/ml Trionyx sinensis Wiegmann Serpentis Fighting cancer club higher than 100 μMs of oleanolic acid, in table 3.
Table 4 Trionyx sinensis Wiegmann Serpentis Fighting cancer club to HCT116 apoptosis enzyme Caspase-3 activity influence (
n=6)
(note: compare * P<0.05 with blank, * * P<0.01; #P<0.05 is compared with oleanolic acid.)
Experiment shows, 125 μ g/ml, 250 μ g/ml, 500 μ g/ml Trionyx sinensis Wiegmann Serpentis Fighting cancer club and 100 μMs of oleanolic acid process HCT116 cell 48h, can significantly improve the activity of HCT116 apoptosis enzyme Caspase-3, and the effect of Trionyx sinensis Wiegmann Serpentis Fighting cancer club shows certain dose dependent.Wherein, the action intensity of 500 μ g/ml Trionyx sinensis Wiegmann Serpentis Fighting cancer club higher than 100 μMs of oleanolic acid, in table 4.
Experimental example 3 Trionyx sinensis Wiegmann Serpentis Fighting cancer club is on the impact of HCT116 cell BCL-2 and BAX protein expression level
Table 5 Trionyx sinensis Wiegmann Serpentis Fighting cancer club on HCT116 cell BCL-2 protein expression level impact (
n=6)
(note: compare * P<0.05 with blank, * * P<0.01; #P<0.05 is compared with oleanolic acid.)
Experiment shows, 250 μ g/ml, 500 μ g/ml Trionyx sinensis Wiegmann Serpentis Fighting cancer club and 100 μMs of oleanolic acid process HCT116 cell 48h, significantly can reduce HCT116 cell BCL-2 protein expression level, and the effect of Trionyx sinensis Wiegmann Serpentis Fighting cancer club shows certain dose dependent.Wherein, the action intensity of 500 μ g/ml Trionyx sinensis Wiegmann Serpentis Fighting cancer club is suitable with 100 μMs of oleanolic acid, in table 5.
Table 6 Trionyx sinensis Wiegmann Serpentis Fighting cancer club on the impact of HCT116 cell BAX protein expression level (
n=6)
(note: compare * P<0.05 with blank, * * P<0.01; #P<0.05 is compared, ##P<0.01 with oleanolic acid.)
Experiment shows, 125 μ g/ml, 250 μ g/ml, 500 μ g/ml Trionyx sinensis Wiegmann Serpentis Fighting cancer club and 100 μMs of oleanolic acid process HCT116 cell 48h, can significantly improve HCT116 cell BAX protein expression level, and the effect of Trionyx sinensis Wiegmann Serpentis Fighting cancer club shows certain dose dependent.Wherein, the action intensity of 500 μ g/ml Trionyx sinensis Wiegmann Serpentis Fighting cancer club is suitable with 100 μMs of oleanolic acid, in table 6.
Experimental example 4 Trionyx sinensis Wiegmann Serpentis Fighting cancer club is on the impact of HCT116 cellular morphology
The results are shown in Figure 1.Wherein, A: blank; B: Trionyx sinensis Wiegmann Serpentis Fighting cancer club (125 μ g/ml); C: Trionyx sinensis Wiegmann Serpentis Fighting cancer club (250 μ g/ml); D: Trionyx sinensis Wiegmann Serpentis Fighting cancer club (500 μ g/ml); E: oleanolic acid (100 μMs)
Experiment shows, 125 μ g/ml, 250 μ g/ml, 500 μ g/ml Trionyx sinensis Wiegmann Serpentis Fighting cancer club and 100 μMs of oleanolic acid can induce HCT116 apoptosis, such that HCT116 cellular morphology reduces, karyon shrinkage, to break, apoptosis morphology change occurs.
To sum up, pharmaceutical composition of the present invention can significantly suppress Human colorectal carcinoma epithelial cell HCT116 to breed, and the Trionyx sinensis Wiegmann Serpentis Fighting cancer club of 500 μ g/ml is suitable with 100 μMs of oleanolic acid action intensities.The anticancer activity that can significantly improve HCT116 apoptosis enzyme Caspase-9, Caspase-3 of Trionyx sinensis Wiegmann Serpentis, reduces the BCL-2 protein expression level of HCT116, improves the BAX protein expression level of HCT116, and apoptosis occurs induction HCT116.
Claims (10)
1. treat a pharmaceutical composition for colorectal cancer, it is characterized in that: it is the preparation be prepared from by the crude drug of following weight proportioning:
Carapax Trionycis 18 parts ~ 22 parts, Herba Hedyotidis Diffusae 18 parts ~ 22 parts, Rhizoma Curcumae 16 parts ~ 20 parts, the Radix Astragali 16 parts ~ 20 parts.
2. pharmaceutical composition as claimed in claim 1, is characterized in that: it is the preparation be prepared from by the crude drug of following weight proportioning: Carapax Trionycis 20 parts, Herba Hedyotidis Diffusae 20 parts, Rhizoma Curcumae 18 parts, the Radix Astragali 18 parts.
3. pharmaceutical composition as claimed in claim 1 or 2, it is characterized in that: it is active component by the protogenic medicinal powder of Carapax Trionycis, Herba Hedyotidis Diffusae, Rhizoma Curcumae, the Radix Astragali, water or extractive with organic solvent, add pharmaceutically acceptable adjuvant or complementary composition is prepared into preparation.
4. pharmaceutical composition as claimed in claim 3, is characterized in that: described preparation is decoction, powder, pill, granule, tablet or oral liquid.
5. prepare a method for the pharmaceutical composition as described in claim 1-4 any one, it is characterized in that: it comprises following steps:
A, get crude drug in proportion, smash;
B, use ethanol extraction, be prepared into extractum;
C, add pharmaceutically conventional adjuvant or complementary composition, be prepared into preparation.
6. preparation method as claimed in claim 5, is characterized in that: described ethanol is the ethanol water of 85%.
7. the pharmaceutical composition described in claim 1-4 any one is preparing the purposes treated and/or prevented in the medicine of colorectal cancer.
8. purposes as claimed in claim 7, is characterized in that: described medicine is the medicine treating and/or preventing one or both cancers in rectal cancer, colon cancer.
9. purposes as claimed in claim 8, is characterized in that: described medicine is the medicine suppressing colon cancer cell HCT116 propagation.
10. purposes as claimed in claim 9, is characterized in that: described medicine is the activity improving in colon cancer cell HCT116 apoptosis enzyme Caspase-9, Caspase-3 one or both; Reduce the BCL-2 protein expression level of colon cancer cell HCT116; Improve the BAX protein expression level of colon cancer cell HCT116; There is apoptotic medicine in induction colon cancer cell HCT116.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113648394A (en) * | 2021-09-14 | 2021-11-16 | 关晓仪 | Anticancer traditional Chinese medicine combination extract, single medicine extract composition and application |
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| CN1286992A (en) * | 2000-09-08 | 2001-03-14 | 沈汉澄 | Medicine for resisting tumor and carcinomatosis |
| KR101442141B1 (en) * | 2014-04-07 | 2014-09-23 | 김태진 | Herbal medicine composition for anti-cancer and forms thereof |
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2015
- 2015-09-30 CN CN201510641656.6A patent/CN105106870A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1286992A (en) * | 2000-09-08 | 2001-03-14 | 沈汉澄 | Medicine for resisting tumor and carcinomatosis |
| KR101442141B1 (en) * | 2014-04-07 | 2014-09-23 | 김태진 | Herbal medicine composition for anti-cancer and forms thereof |
Non-Patent Citations (1)
| Title |
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| 张丽红等: "益肠泰诱导CCL229人大肠癌细胞凋亡的形态学研究", 《中国基层医药》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113648394A (en) * | 2021-09-14 | 2021-11-16 | 关晓仪 | Anticancer traditional Chinese medicine combination extract, single medicine extract composition and application |
| CN113648394B (en) * | 2021-09-14 | 2022-08-02 | 关晓仪 | Anticancer traditional Chinese medicine combination extract, single medicine extract composition and application |
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Application publication date: 20151202 |