CN101229214A - Medical applications of stringy stonecrop ethamol soluble extractive - Google Patents
Medical applications of stringy stonecrop ethamol soluble extractive Download PDFInfo
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- CN101229214A CN101229214A CNA2008100192006A CN200810019200A CN101229214A CN 101229214 A CN101229214 A CN 101229214A CN A2008100192006 A CNA2008100192006 A CN A2008100192006A CN 200810019200 A CN200810019200 A CN 200810019200A CN 101229214 A CN101229214 A CN 101229214A
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Abstract
The invention relates to a new usage of sedum sarmentosun bunge extract, that is, an application of the sedum sarmentosun bunge in preparing anti-cancer medicine. The invention develops new medical usage of the extract of the sedum sarmentosun bunge and exploits a new application field. The extract of the sedum sarmentosun bunge has significant effect of inhibiting the proliferation of hepatoma cells, regulating and controlling cells period, and inhibiting relevant gene expression in RNA and protein level, so the invention can be used for preparing anti-tumor medicine.
Description
Technical field
The present invention relates to a kind of medical applications of Chinese herbal medicine extract, relate in particular to a kind of medical applications of Herba Sedi ethanol extract.
Background technology
Herba Sedi (Sedum sarmentosum Bunge) has another name called crouch stem Herba hylotelephii erythrosticti, half of Mus tooth, Semen Benincasae grass, Herba Sedi Multicaulis, Herba Sedi Sarmentosi, is used as medicine and begins to be stated from " BAICAO mirror ".It is the perennial meat woody plant of Crassulaceae.Herba Sedi is to go through version " one of conventional Chinese medicine that Chinese pharmacopoeia is recorded.Herba Sedi is a kind of important resources of medicinal plant, gathers at summer, Qiu Erji, removes impurity, its fresh or dry all herbal medicine, effect with heat-clearing and toxic substances removing, inducing diuresis to remove edema, evacuation of pus granulation promoting is used for damp and hot yellow cellulitis, erysipelas ulcer; dysuria; the carbuncle skin infection, the treatment of acute and chronic hepatitis, and have preferably hepatoprotective, immunosuppressant, estrogen action, ace inhibiting effect and effect such as antibacterial [Pan Jinhuo; Chinese Pharmaceutical, 16:365-366 (2002); Kim WH, et al.J Nutr Sci Vitaminol, 50:100-105 (2004); Oh H, et al.Biol Pharm Bull.27:2035-2037 (2004)], be medicinal plants with bright prospects.Among the people have special efficacy with Herba Sedi treatment acute hepatitis, and the single Herba Sedi is generally taken and can cure in 15 days, and the effect of performance is jaundice eliminating, falls enzyme when treatment, and liver is swollen improves, and dull pain in liver disappears, and urine transfers to normal.
According to relevant bibliographical information, Herba Sedi can effectively suppress inflammatory exudation, reduce hepatocyte injury, obviously reduce NSC 334200 transaminase (ALT), effect of reducing enzyme levels is [Guo Jia etc. rapidly and lastingly, the Shanghai medical magazine, 11:47 (1991)], this medicine that this and history records are put down in writing has the effect of " heat-clearing and toxic substances removing, detumescence " to fit like a glove.The main component of Herba Sedi is (He Aimin etc., Chinese herbal medicine, 28:517-522 (1997) such as sarmentosin, sterols material, Flavonoid substances, some glucide and alkaloids substance; He Aimin etc., China Medicine University's journal, 28:271-274 (1997); He Aimin, et al.Phytochemistry, 49:2607-2610 (1998)].Studies show that flavone compound suppresses the initial of lipid peroxidation by removing superoxide anion and hydroxy radical, is the key structure of removing oxygen-derived free radicals, is to promote fatigue recovery, increases the effective ingredient of body movement ability.Herba Sedi has been widely used in treating laryngopharynx swelling and pain, pyretic stranguria burn due to hot liquid or fire, snake bite and insect sting and various acute and chronic active hepatitis clinically, but does not appear in the newspapers as yet for Herba Sedi ethanol extract anti-tumor activity and Its Mechanisms thereof.
Summary of the invention
Goal of the invention: the new medical applications that the object of the present invention is to provide the Herba Sedi ethanol extract.
Technical scheme: Herba Sedi ethanol extract of the present invention prepares as follows:
With the Herba Sedi Radix Glycyrrhizae with 95% ethanol extraction 2～3 times, the ethanol consumption be 10 milliliters/(gram Radix Glycyrrhizae * time), merge extractive liquid,, concentrate, extract concentrated solution adopts petroleum ether extraction to remove lipid, and the petroleum ether consumption is 2 a milliliters/gram Radix Glycyrrhizae, fling to petroleum ether, reuse dissolve with methanol, methanol usage are 0.2 milliliter/gram Radix Glycyrrhizae, evaporated under reduced pressure.
General flavone content is 4～30% in the Herba Sedi extract that makes by above-mentioned preparation method, and sterol content is 3.5～20%, and sarmentosin content is 0.4～10%, triterpenes 0.2～10%.
The Herba Sedi ethanol extract shows the influence experiment of hepatoma carcinoma cell HepG2 propagation, 200 μ g/ml Herba Sedi ethanol extract promptly have significant inhibitory effect to hepatoma carcinoma cell in effect after 24 hours, and effect after 72 hours each dosage group ethanol extract all hepatoma carcinoma cell is had the inhibitory action of highly significant.Illustrate that the Herba Sedi ethanol extract has the obvious suppression effect to liver cancer cell growth, and certain dose-dependence is arranged.
Flow cytometry shows that the Herba Sedi ethanol extract can make hepatoma carcinoma cell be blocked in static (G0/G1) phase, thereby has suppressed the division growth of cell, has notable antitumor activity.
Detect of the influence of Herba Sedi ethanol extract by investigating immunocytochemistry, find that the Herba Sedi ethanol extract can significantly suppress the expression of the two, especially has the inhibitory action of highly significant to VEGF the expression of HepG2VEGF and c-myc.Explanation thus, the Herba Sedi ethanol extract has inhibitory action to hepatoma carcinoma cell.
In addition, detect of the influence of Herba Sedi ethanol extract, find that ethanol extract can significantly suppress the mRNA level of VEGF, c-myc, confirms that further the Herba Sedi ethanol extract has inhibitory action to hepatoma carcinoma cell the mRNA level of HepG2VEGF and c-myc by investigating the RT-PCR method.
To sum up research, the Herba Sedi ethanol extract can be used for preparing medicines resistant to liver cancer.
Beneficial effect: the present invention has excavated new medical application to the Herba Sedi ethanol extract, has opened up a new application.The Herba Sedi ethanol extract has the effect of significant inhibition hepatoma cell proliferation, regulates cell cycle, suppresses the tumor-related gene expression from RNA and protein level, can be used as medicines resistant to liver cancer.
Description of drawings
Fig. 1 is to the influence of its cell cycle behind the Herba Sedi ethanol extract effect HepG2 cell 48h.The cell cycle distribution of HepG2 when A is 0 μ g/ml for the medicine final concentration; The HepG2 cell cycle changed when B was 50 μ g/ml for the medicine final concentration; The HepG2 cell cycle changed when C was 100 μ g/ml for the medicine final concentration; The HepG2 cell cycle changed when D was 200 μ g/ml for the medicine final concentration.Increase with drug level, cell blocks in the quantity of resting stage and increases gradually.
Fig. 2 is the influence that the Herba Sedi ethanol extract is expressed HepG2 cell VEGE, c-myc.A, B, C, D, E are respectively medicine final concentration 0,25,50,100, the expression of VEGF during 200 μ g/ml; F, G, H, I, J are respectively medicine final concentration 0,25,50,100, the expression of c-myc during 200 μ g/ml.Increase with drug level, VEGF, c-myc expression descend gradually.
Fig. 3 is a Herba Sedi second ethanol extract to the electrophoretic analysis figure of the mRNA level affects of the VEGF of HepG2 cell and c-myc.Increase with drug level, VEGF and c-myc mRNA level are significantly reduced.
The specific embodiment
Embodiment 1: the preparation of Herba Sedi ethanol extract
Herba Sedi Radix Glycyrrhizae 500g soaked 5 days in 95% ethanol 5000mL, and filtered through gauze is collected medicinal liquid.Repeat 2 times, merge medicinal liquid, the centrifuging and taking supernatant, the gained supernatant is flung to solvent through reduction vaporization, removes lipid with petroleum ether 1000mL extraction, flings to petroleum ether, reuse 100mL dissolve with methanol, evaporated under reduced pressure, the dry ethanol extract that gets.
Embodiment 2: ethanol extract is to the inhibitory action of hepatoma cell proliferation
Ethanol extract dilutes through the DMSO dissolving and with the RPMI1640 culture medium, and making the DMSO final concentration is 0.1%, standby through the sterile filters degerming.Human hepatoma cell strain HepG2 places 37 ℃, 5%CO with the RPMI1640 culture fluid that contains 10% calf serum
2Cultivate in the incubator.To exponential phase, adjusting cell concentration is 1 * 10 with tumor cell culture
4Individual/ml, be inoculated in 96 orifice plates, 100 μ l/ holes, 4 hours adherent after, adding ethanol extract makes final concentration be respectively 25,50,100,200 μ g/ml's, other establishes negative control group (RPMI1640 culture medium) and 0.1%DMSO solvent control group, establish 6 parallel holes for every group, continue to cultivate 24,48,72 hours, mtt assay detects the propagation result.
Each dosage group sees Table 1 to the exercising result of HepG2.The result shows that 200 μ g/ml ethanol extract promptly have significant inhibitory effect to hepatoma carcinoma cell in effect after 24 hours, and effect after 72 hours each dosage group ethanol extract all hepatoma carcinoma cell is had the inhibitory action of highly significant.Illustrate that the Herba Sedi ethanol extract has the obvious suppression effect to liver cancer cell growth, and certain dose-dependence is arranged.
Table 1 Herba Sedi ethanol extract is to the influence of hepatoma carcinoma cell HepG2 propagation
| Dosage (μ g/ml) | 24h | 48h | 72h |
| 0 25 50 100 200 | 0.3397±0.0308 0.3138±0.0257 0.3104±0.0087 0.3010±0.0038 0.2978±0.0205 * | 0.5167±0.0356 0.5108±0.0315 0.5080±0.0170 0.4976±0.0312 0.4458±0.0452 * | 0.8394±0.0261 0.7455±0.0208 *** 0.6823±0.0256 *** 0.6764±0.0609 *** 0.6738±0.0368 *** |
*P＜0.05,
* *Compare with matched group in p＜0.001
Embodiment 3: the Flow cytometry cell cycle:
Collect the HepG2 cell 1 * 10 behind each dosage effect 48h of matched group and ethanol extract respectively
6Individual, PI dyeing is carried out cell cycle analysis with flow cytometry, the results are shown in accompanying drawing 1.Flow cytometry shows that ethanol extract can make hepatoma carcinoma cell block the phase in G0/G1, thereby has suppressed its proliferation activity.
Embodiment 4: immunocytochemistry detects tumor correlated albumen and expresses:
The take the logarithm HepG2 cell of trophophase is with 5 * 10
4Individual/ml cell concentration is inoculated in six orifice plates that are placed with coverslip, after 4h is adherent medicine is added wherein with variable concentrations.Take out coverslip after cultivating 48h.PBS cleans specimen 2 times, and 4 ℃ of 80% propanol are fixing, the strict SABC that presses of staining procedure, the operation of DAB test kit description.Detect VEGF and c-Myc protein expression.Establish matched group simultaneously.Coloration result the positive cell of cell that pale brown color exceeds background occurs with cytoplasm or nuclear membrane.Without the matched group VEGF of ethanol extract effect (Fig. 2, A) and c-myc (Fig. 2 F) has very strong positive expression.25,50, the vegf expression level of 100 and 200 μ g/ml ethanol extract effects hepatoma carcinoma cell after 48 hours according to concentration increase progressively be highly significant descend (Fig. 2, B, C, D, E); C-myc express and also to significantly decrease (Fig. 2, G, H, I, J).
Calculate every group positive cell rate.Analysis result sees Table 2.Ethanol extract can significantly suppress the expression of the two.Explanation thus, the Herba Sedi ethanol extract has significant inhibitory effect to the expression of hepatoma carcinoma cell tumor-related gene.
Table 2 Herba Sedi ethanol extract is to the influence of HepG2VEGF and c-myc protein expression level
| Dosage (μ g/ml) | N | VEGF | c-myc |
| 0 25 50 100 200 | 10 10 10 10 10 | 0.3507±0.0419 0.2805±0.0277 *** 0.2465±0.0337 *** 0.2050±0.0171 *** 0.1448±0.0283 *** | 0.4321±0.0837 0.3703±0.0874 0.3259±0.0841 * 0.2867±0.0565 *** 0.2799±0.0334 *** |
*P＜0.05,
* *Compare with matched group p＜0.001
Embodiment 5:RT-PCR detects the change of the mRNA level of VEGF, c-myc
Experimental group and matched group HepG2 cell extract RNA with Biozol reagent respectively, obtain white precipitate after extracting the operation of test kit step, add 1 ‰ DEPC water, 30 μ l to RNA and dissolve fully.The concentration of determined by ultraviolet spectrophotometry RNA and purity.Getting and respectively organize each 2 μ g of total RNA and carry out the synthetic cDNA of reverse transcription respectively, is that interior mark carries out sxemiquantitative RT-PCR with β-actin.At 94 ℃ of pre-degeneration 5min, be respectively 94 ℃ of degeneration 30s then earlier, 58 ℃ of annealing 40s, 72 ℃ are extended 30s, totally 30 circulations, last 72 ℃ are extended 7min.1.5% agarose gel electrophoresis, the gel imaging system analysis result.Ethanol extract sees Table 3 and Fig. 3 to the influence of hepatoma carcinoma cell VEGF, c-myc expression intensity.Ethanol extract can significantly suppress the mRNA level of VEGF, c-myc.Confirm that further the Herba Sedi ethanol extract has inhibitory action to hepatoma carcinoma cell.
Table 3 Herba Sedi ethanol extract is to the inhibitory action of the mRNA transcriptional level of HepG2VEGF and c-myc
| Dosage (μ g/ml) | The suppression ratio of VEGF (%) | The suppression ratio of c-myc (%) |
| 25 50 100 200 | 21.62 39.53 70.78 90.41 | 1.87 42.03 64.11 82.41 |
Claims (6)
1. the application of Herba Sedi ethanol extract in the preparation medicines resistant to liver cancer.
2. application according to claim 1, it is characterized in that described Herba Sedi ethanol extract prepares as follows: with the Herba Sedi Radix Glycyrrhizae with 95% ethanol extraction 2～3 times, the ethanol consumption be 10 milliliters/(gram Radix Glycyrrhizae * time), merge extractive liquid,, concentrated, extract concentrated solution adopts petroleum ether extraction to remove lipid, the petroleum ether consumption is 2 a milliliters/gram Radix Glycyrrhizae, flings to petroleum ether, the reuse dissolve with methanol, methanol usage is 0.2 a milliliter/gram Radix Glycyrrhizae, evaporated under reduced pressure.
3. application according to claim 1 is characterized in that the application of Herba Sedi ethanol extract in the propagation that suppresses hepatoma carcinoma cell.
4. application according to claim 1 is characterized in that the Herba Sedi ethanol extract is blocked resting stage in hepatoma carcinoma cell, the application in the division growth of inhibition cell.
5. application according to claim 1 is characterized in that the Herba Sedi ethanol extract at VEGF that reduces tumor-related gene and the protein expression level of c-myc, the application from protein level inhibition hepatoma cell proliferation.
6. application according to claim 1 is characterized in that the Herba Sedi ethanol extract in VEGF that reduces tumor-related gene and the mRNA level of c-myc, the application from the propagation of transcriptional level inhibition hepatoma carcinoma cell.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2012154799A1 (en) * | 2011-05-09 | 2012-11-15 | Sheng Yu-Hwa Peter | Herbal composition for treating cancer |
| CN111297935A (en) * | 2019-12-20 | 2020-06-19 | 河南省医药科学研究院 | Sedum sarmentosum extract and preparation method and application thereof |
-
2008
- 2008-01-16 CN CNA2008100192006A patent/CN101229214A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2012154799A1 (en) * | 2011-05-09 | 2012-11-15 | Sheng Yu-Hwa Peter | Herbal composition for treating cancer |
| JP2014513691A (en) * | 2011-05-09 | 2014-06-05 | ユウ−ファ ピーター シェン， | Herbal composition for treating cancer |
| CN111297935A (en) * | 2019-12-20 | 2020-06-19 | 河南省医药科学研究院 | Sedum sarmentosum extract and preparation method and application thereof |
| CN111297935B (en) * | 2019-12-20 | 2021-10-01 | 河南省医药科学研究院 | Sedum sarmentosum extract and preparation method and application thereof |
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Open date: 20080730 |