CN103006741A - Semen lepidii anti-tumor effective component extract, as well as preparation method and application thereof - Google Patents
Semen lepidii anti-tumor effective component extract, as well as preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a semen lepidii anti-tumor effective component, a preparation method of the semen lepidii anti-tumor effective component and an application of the semen lepidii anti-tumor effective component in the field of anti-tumor. The preparation method comprises the following steps of: extracting semen lepidii (roasted) with water; and extracting with ethyl acetate and water saturated n-butyl alcohol sequentially; collecting water saturated n-butyl alcohol extract; separating with a silica (200 to 300 meshes) column; and performing gradient elution with dichloromethane-methanol in different proportions, wherein the dichloromethane-methanol (5:1) elution part is a semen lepidii anti-tumor effective part. The part has a remarkable effect of inhibiting U251 (glioma cell line), HepG2 (human hepatoma cell line), SGC (human gastric carcinoma cell), mcf-7 (human breast cancer cell) and SCLC (lung carcinoma cell), and can be used for preparing an anti-tumor medicament.
Description
Technical field
The present invention relates to lepidii,semen anti-cancer effective component extract and pharmaceutical composition thereof.
Background technology
At present because the aggravation of environmental pollution, the quickening of rhythm of life, the increase of life stress, the incidence rate of cancer is in the trend of rising, show that according to World Health Organization's latest data before the year two thousand twenty, global cancer morbidity will increase by 50%, China's cancer morbidity then increased with the speed in every year 2.5% in recent years, seemed more urgent so accelerate the research of cancer therapy drug.
Anti-tumor drug is mainly chemical synthetic drug clinically at present, though the treatment of tumor disease is improved greatly, these antineoplaston chemicals ubiquity toxic and side effects are large, easily produce the shortcomings such as drug resistance.By comparison, the untoward reaction less that Chinese medicine shows clinically, medical expense is relatively low, and can keep the seriality of medication, aspect prevention and the treatment tumor obvious advantage is being arranged.From the 1950's, engender the bibliographical information of Chinese medicine prevention tumor, the drug study research of standard, Chinese medicine begins to be widely used in the control of malignant tumor.In recent years, both at home and abroad the research that derives from natural antitumor drug is paid much attention to, existing natural resources of Chinese medicinal materials has been carried out inside and outside antitumor screening test research, experimental result shows that a lot of Chinese medicines have antitumor action in various degree.
Semen Lepidii (Semen Descurainiae) (
Semen Descurainiae.Semen Lepidii) be Cruciferae (
Cruciferae) two kinds
Original plant, namely Lepidium sativum L. (
Lepidium apetalum Wild) and descurainia sophia (l.) webb ex prantl (
Descurainia Sophia L.Webb ex Prantl) dry mature seed.The former practises and to be called lepidii,semen, mainly is distributed in the ground such as northeast, Hebei, Shandong, Shanxi, Sichuan, the Inner Mongol of China; The latter practises and to be called Semen Descurainiae or East China Semen Lepidii (Semen Descurainiae), mainly is distributed in the ground such as northeast, Henan, Shandong, Zhejiang, Gansu, the Inner Mongol of China.Be mainly used in clinically treating sputum stop up lung, dyspnea and cough with excessive sputum, fullness and distention in the chest and hypochondrium, must not put down sleeping, ascites pleural fluid swollen, dysuria, pulmonary heart disease heart failure etc. (Ministry of Health of the People's Republic of China's committee of pharmacopeia. " Chinese pharmacopoeia. Beijing: Chemical Industry Press, 2010.).
Modern pharmacological research shows, Semen Lepidii (Semen Descurainiae) have relieving cough and asthma, heart tonifying, diuresis, antibiotic, anticancer (medicine management Chinese herbal medicine information center of country station. active ingredient of autonomic drug handbook: 119,411,556-557,962-963,998.), transfer blood fat (Liu is faithful and upright. the pharmacy practice magazine, 2000,18(1): the effect such as 15-17.).Wherein Semen Sinapis Albae is the cough-relieving effective ingredient, benzyl mustard oil has broad-spectrum antibacterial action, and its heart tonifying, diuresis are main and its cardiac glycoside that contains: the relevant (Chen Yuqun of strophanthus aglucon (strophanthidine), evobioside (evobioside), evomonoside (evomonoside), Semen Lepidii glycosides (helveticoside) and erysimin (erysimoside), Li Rongzhi, Wang Yunwen. Acta Pharmaceutica Sinica, 1981,16(1): 62-64. C.A.:1974:81,166395.).Semen Lepidii (Semen Descurainiae) has extremely strong inhibitory action to KB cell and thousand field cervical cancer cell strains, and the cancerous cell of Ehrlich ascites carcinoma is had obvious inhibitory action, and almost non-toxic side reaction (Chang Minyi. Chinese medicine medicine for preventing: 482-483; Hunan science tech publishing house .).Sun Kai research finds that the strophanthus aglucon has obvious inhibitory action, ED to human nasal pharyngeal cancer (9KB) cell
50﹤ 1 μ g/ml(Sun Kai. the chemical constitution study of Semen Descurainiae [D]. Shenyang Pharmaceutical University, 2005).Chinese patent 200810226062.9 " Semen Lepidii extract (Semen Descurainiae extract) is in the application of preparation in the antitumor drug " has related to the extraction solvent of Semen Lepidii extract (Semen Descurainiae extract) and effect but has not related to the concrete effective site of Semen Lepidii (Semen Descurainiae) antitumor action and the detailed name of an article.
Summary of the invention
One of purpose of the present invention is that lepidii,semen (stir-fry) anti-cancer effective component extract and preparation method thereof is provided.
Two of purpose of the present invention is that the application of said active component extract in preparation antitumor and related drugs is provided.
Technical scheme of the present invention is as follows:
Lepidii,semen anti-cancer effective component extract, it is after being pulverized by the lepidii,semen of parch, water is in 90-100 ℃ of heating extraction, extracting solution is concentrated, precipitate with ethanol, the centrifugal precipitation of removing, concentrating under reduced pressure is removed ethanol, extract successively with ethyl acetate and water-saturated n-butanol, collect the water-saturated n-butanol position, cross silicagel column, carry out gradient elution with the methylene chloride-methanol system, collecting dichloromethane and methanol volume ratio is the eluent of 5:1, boils off the lepidii,semen anti-cancer effective component extract that solvent obtains.
A kind of method for preparing above-mentioned lepidii,semen anti-cancer effective component extract, it comprises the steps:
Step 1. is pulverized the lepidii,semen of parch, the water reflux, extract,, and cooling, centrifugal, concentrated extracting solution carries out precipitate with ethanol with 95% ethanol, and the centrifugal precipitation of removing concentrates and removes ethanol, gets extracting solution;
Step 2. extracts the extracting solution of step 1 gained successively with ethyl acetate, water saturated butanol solution, collect the water-saturated n-butanol extract, carry out chromatography with silicagel column, eluant is the methylene chloride-methanol of different proportion, collecting dichloromethane and methanol volume ratio is the eluent of 5:1 ratio, rotary evaporation namely gets lepidii,semen anti-cancer effective component extract after reclaiming organic solvent.
The present invention further provides the application of lepidii,semen anti-cancer effective component extract in the preparation antitumor drug.To suppress U251, HepG
2, SGC, mcf-7, SCLC multiplication capacity be that activity index carries out the anti-tumor activity experiment.Experimental result shows, in the experiment of cell in vitro poison, can obviously suppress U251, HepG
2, SGC, mcf-7, SCLC propagation, have cytotoxicity, can be used for preparing the antineoplaston medicine.
Beneficial effect
1, advantage of the present invention and effect are to adopt the conventional methods such as solvent extraction, extraction, extract from lepidii,semen and have obtained the new effective site with antitumor action, and this effective site the experiment proved that to have vitro inhibition U251, HepG
2, SGC, mcf-7, SCLC growth activity.
2, the present invention has expanded the raw material sources of antitumor drug, has enlarged the purposes of lepidii,semen, makes lepidii,semen develop into the new raw material of antitumor drug, can significantly improve the added value of Semen Lepidii (Semen Descurainiae).
The specific embodiment
Below listed embodiment help those skilled in the art to understand better the present invention, but do not limit the present invention in any way.
The preparation of embodiment 1. lepidii,semen anti-cancer effective component extracts
Chinese crude drug lepidii,semen (stir-fry) is pulverized, water reflux, extract, 3h with 10 times of quality, residue after centrifugal repeats to extract 1 time, centrifugal, merge all aqueous extracts, concentrated extracting solution adds 95% ethanol and carries out precipitate with ethanol to 80% volume (ml) of crude drug mass number (gram), and the concentration of ethanol is about 60% ~ 70% in the medicinal liquid, hold over night, centrifugal, discard precipitation, decompression recycling ethanol, add isopyknic ethyl acetate extraction to organic facies near colourless, remaining water liquid is with water saturated n-butanol extraction 6 times, and the recovery n-butyl alcohol gets the water-saturated n-butanol extract.Get the saturated n-butanol extract of suitable quantity of water and cross silicagel column, methylene chloride-methanol (100:0,50:1,20:1 with the different volumes ratio, 10:1,5:1,1:1 and 0:100) carry out gradient elution, the used effluent volume of each gradient is 4 times of column volumes, collected volume is than being the methylene chloride-methanol eluent of 5:1, concentrated, vacuum drying namely gets lepidii,semen anti-cancer effective component extract.
Get lepidii,semen anti-cancer effective component extract and carry out efficient liquid phase chromatographic analysis.Actual conditions is: chromatographic column is that (4.6 * 150mm), flow velocity is 1.0ml/min to Shimadzu shimpack C18 post.Gradient elution: A phase: 0.01% formic acid-water; The B phase: methanol, elution program are 0.0min～45.00min:B:2%～50%.Lepidii,semen anti-cancer effective component extract mainly occurs 5 and keeps the peak under this chromatographic condition, and retention time is respectively: t
1=2.8min, t
2=9.6min, t
3=30.4min, t
4=39.6min, t
3=41.2min.
The preparation of embodiment 2. lepidii,semen anti-cancer effective component extracts
Lepidii,semen (stir-fry) is pulverized, water reflux, extract, 2h with 8 times of quality, residue after centrifugal repeats to extract 2 times, centrifugal, merge all aqueous extracts, concentrated extracting solution is to the volume (ml) of crude drug mass number (gram), add 95% ethanol and carry out precipitate with ethanol, the concentration of ethanol is about 60% in the medicinal liquid, hold over night, centrifugal, discard precipitation, decompression recycling ethanol, add isopyknic ethyl acetate extraction to organic facies near colourless, remaining water liquid with n-butanol extraction to organic facies near colourless, the collection n-butanol portion.Get an amount of n-butanol extract and cross silicagel column, methylene chloride-methanol (20:1 in varing proportions, 10:1,5:1,1:1) carry out gradient elution, the used effluent volume of each gradient is 4 times of column volumes, collect the eluent of 5:1, concentrated, vacuum drying namely gets lepidii,semen anti-cancer effective component extract.
Get lepidii,semen anti-cancer effective component extract and carry out efficient liquid phase chromatographic analysis, chromatographic condition is with embodiment 1.The result shows that the reservation situation of lepidii,semen anti-cancer effective component extract on high performance liquid chromatography is consistent with embodiment's 1.
The preparation of embodiment 3. lepidii,semen anti-cancer effective component extracts
Lepidii,semen (stir-fry) is pulverized, water reflux, extract, 2h with 8 times of quality, residue after centrifugal repeats to extract 2 times, centrifugal, merge all aqueous extracts, concentrated extracting solution is to the volume (ml) of crude drug mass number (gram), add 95% ethanol and carry out precipitate with ethanol, the concentration of ethanol is about 60% in the medicinal liquid, hold over night, centrifugal, discard precipitation, decompression recycling ethanol, add isopyknic ethyl acetate extraction to organic facies near colourless, remaining water liquid with n-butanol extraction to organic facies near colourless, the collection n-butanol portion.Get an amount of n-butanol extract and cross silicagel column, methylene chloride-methanol (100:0 in varing proportions, 10:1,5:1,1:1) carry out gradient elution, the used effluent volume of each gradient is 4 times of column volumes, collect the eluent of 5:1, concentrated, vacuum drying namely gets lepidii,semen anti-cancer effective component extract.
Get lepidii,semen anti-cancer effective component extract and carry out efficient liquid phase chromatographic analysis, chromatographic condition is with embodiment 1.The result shows that the reservation situation of lepidii,semen anti-cancer effective component extract on high performance liquid chromatography is consistent with embodiment's 1.
The experiment of embodiment 4. anti-tumor activities
The lepidii,semen effective site that obtains take embodiment 1 is as tested medicine, In vitro culture U251, HepG
2, SGC, mcf-7, SCLC, fetching is counted the cell of trophophase, after trypsinization, 1500r ﹒ min
-1Centrifugal 5min is resuspended in cell in the culture medium, and cell concentration transfers to 2 * 10
4Individual/ml, be inoculated in the 96 porocyte culture plates, every hole 100 μ l, the placement cell culture incubator (37 ℃, 5%CO
2) the middle 24h that cultivates, adding afterwards tested medicine 20 μ l, it is 0.5%DMSO that negative control group adds final concentration, each group is all established 3 again holes.Behind dosing 48h, every hole adds 5mg ﹒ ml
-1MTT reagent 20 μ l continue to place 37 ℃ to cultivate 4h.Every hole adds DMSO100 μ l, and the vibration mixing is measured each hole at the absorbance at wavelength 570nm place.Calculate cell proliferation inhibition rate: suppression ratio (%)=(1-experimental group absorbance/matched group absorbance) * 100%.
Table 1 lepidii,semen extract is to the inhibitory action of U251
Table 2 lepidii,semen extract is to the suppression ratio (%) of human tumor cells
| Tested material | Concentration (μ g/ml) | HepG 2 | mcf-7 | SCLC | SGC |
| The lepidii,semen extract | 5 | 80.66 | 80.81 | 48.86 | 68.56 |
| 5-fluorouracil | 5 | 66.48 | 41.54 | 55.68 | 48.66 |
The lepidii,semen anti-cancer effective component extract repetition present embodiment that obtains with embodiment 2-3 obtains identical result.
Claims (4)
1. lepidii,semen anti-cancer effective component extract, it is characterized in that: it is after being pulverized by the lepidii,semen of parch, water is in 90-100 ℃ of heating extraction, extracting solution is concentrated, precipitate with ethanol, the centrifugal precipitation of removing, concentrating under reduced pressure is removed ethanol, extract successively with ethyl acetate and water-saturated n-butanol, collect the water-saturated n-butanol position, cross silicagel column, carry out gradient elution with the methylene chloride-methanol system, collecting dichloromethane and methanol volume ratio is the eluent of 5:1, boils off solvent, namely gets lepidii,semen anti-cancer effective component extract.
2. a method for preparing above-mentioned lepidii,semen anti-cancer effective component extract is characterized in that it comprises the steps:
Step 1. is pulverized the lepidii,semen of parch, the water reflux, extract,, and cooling, centrifugal, concentrated extracting solution carries out precipitate with ethanol with 95% ethanol, and the centrifugal precipitation of removing concentrates and removes ethanol, gets extracting solution;
Step 2. extracts the extracting solution of step 1 gained successively with ethyl acetate, water saturated butanol solution, collect the water-saturated n-butanol extract, carry out chromatography with silicagel column, eluant is the methylene chloride-methanol of different proportion, collecting dichloromethane and methanol volume ratio is the eluent of 5: 1 ratios, rotary evaporation namely gets lepidii,semen anti-tumor effective component extract after reclaiming organic solvent.
3. the application of lepidii,semen anti-cancer effective component extract according to claim 1 in the preparation antitumor drug.
4. application according to claim 3 is characterized in that: described antitumor drug is anti-U251, HepG
2, SGC, mcf-7 or SCLC tumor cell medicine.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105168301A (en) * | 2015-10-23 | 2015-12-23 | 河南中医学院 | Application of semen lepidii water extract in preparation of estrogen medicines |
| CN106928287A (en) * | 2017-03-02 | 2017-07-07 | 河南中医药大学 | A kind of method that phenolic glycoside compound is extracted from lepidii,semen and its application |
| CN107184632A (en) * | 2017-07-25 | 2017-09-22 | 北京健旭康技术有限公司 | The effect of beneficial liver and its application of separate row Vegetable spp plant |
| CN107501355A (en) * | 2017-09-06 | 2017-12-22 | 河南中医药大学 | A kind of new glycosides D of northern Roripa and preparation method and application |
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| CN101390919A (en) * | 2008-11-05 | 2009-03-25 | 中国科学院化学研究所 | Use of pepperweed seed extract in preparing anti-tumor medicine |
| CN101779830A (en) * | 2009-01-20 | 2010-07-21 | 河南中烟工业有限责任公司 | Pepperweed seed extract and application thereof in tobacco |
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Patent Citations (2)
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| CN101390919A (en) * | 2008-11-05 | 2009-03-25 | 中国科学院化学研究所 | Use of pepperweed seed extract in preparing anti-tumor medicine |
| CN101779830A (en) * | 2009-01-20 | 2010-07-21 | 河南中烟工业有限责任公司 | Pepperweed seed extract and application thereof in tobacco |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105168301A (en) * | 2015-10-23 | 2015-12-23 | 河南中医学院 | Application of semen lepidii water extract in preparation of estrogen medicines |
| CN105168301B (en) * | 2015-10-23 | 2019-01-25 | 河南中医学院 | Application of the lepidium seed water extract in preparation estrogen drugs |
| CN106928287A (en) * | 2017-03-02 | 2017-07-07 | 河南中医药大学 | A kind of method that phenolic glycoside compound is extracted from lepidii,semen and its application |
| CN106928287B (en) * | 2017-03-02 | 2019-05-17 | 河南中医药大学 | A kind of method and its application for extracting phenolic glycoside compound from lepidii,semen |
| CN107184632A (en) * | 2017-07-25 | 2017-09-22 | 北京健旭康技术有限公司 | The effect of beneficial liver and its application of separate row Vegetable spp plant |
| CN107501355A (en) * | 2017-09-06 | 2017-12-22 | 河南中医药大学 | A kind of new glycosides D of northern Roripa and preparation method and application |
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