CN103006741B - 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 PDF

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CN103006741B
CN103006741B CN201210467137.9A CN201210467137A CN103006741B CN 103006741 B CN103006741 B CN 103006741B CN 201210467137 A CN201210467137 A CN 201210467137A CN 103006741 B CN103006741 B CN 103006741B
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semen
lepidii
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water
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CN103006741A (en
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余江南
奚超群
徐希明
童姗姗
易承学
曹霞
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Jiangsu University
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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

Lepidii,semen anti-cancer effective component extract and method for making thereof and purposes
Technical field
The present invention relates to lepidii,semen anti-cancer effective component extract and pharmaceutical composition thereof.
Background technology
The current aggravation due to environmental pollution, the quickening of rhythm of life, the increase of life stress, the trend of the incidence rate of cancer in rising, according to World Health Organization's latest data, show, before the year two thousand twenty, global cancer morbidity will increase by 50%, China's cancer morbidity increased with annual 2.5% speed in recent years, so accelerate the research of cancer therapy drug, seemed more urgent.
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 that Chinese medicine showed is clinically relatively little, and medical expense is relatively low, and can keep the seriality of medication, aspect prevention and treatment tumor, is having obvious advantage.From the 1950's, engender the bibliographical information of Chinese medicine prevention tumor, the drug study research of standard, Chinese medicine starts 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 to inside and outside antitumor screening test research, experimental result demonstration, a lot of Chinese medicine has antitumor action in various degree.
Semen Lepidii (Semen Descurainiae) ( semen Descurainiae.Semen Lepidii) be Cruciferae ( cruciferae) two kinds
Original plant, 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 is called lepidii,semen, is mainly distributed in the ground such as northeast, Hebei, Shandong, Shanxi, Sichuan, the Inner Mongol of China; The latter practises and is called Semen Descurainiae or East China Semen Lepidii (Semen Descurainiae), is mainly distributed in the ground such as northeast, Henan, Shandong, Zhejiang, Gansu, the Inner Mongol of China.Be mainly used in clinically treating that sputum is stopped up lung, dyspnea and cough with excessive sputum, fullness and distention in the chest and hypochondrium, must not be put down that sleeping, ascites pleural fluid is swollen, dysuria, pulmonary heart disease heart failure etc. (committee of pharmacopeia of Ministry of Health of the People's Republic of China. < < Chinese Pharmacopoeia > >. Beijing: Chemical Industry Press, 2010.).
Modern pharmacological research shows, Semen Lepidii (Semen Descurainiae) have relieving cough and asthma, heart tonifying, diuresis, antibacterial, anticancer (medicine management Chinese herbal medicine information center of country station. active ingredient of autonomic drug handbook: 119,411,556-557,962-963,998.), adjust blood fat (Liu faithful and upright person. pharmacy practice magazine, 2000,18(1): 15-17.) effect such as.Wherein Semen Sinapis Albae is cough-relieving effective ingredient, benzyl mustard oil has broad-spectrum antibacterial action, and the cardiac glycoside that its heart tonifying, diuresis mainly contain with it: 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 to 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 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 [D] of Semen Descurainiae. Shenyang Pharmaceutical University, 2005).Chinese patent 200810226062.9 < < Semen Lepidii extract (Semen Descurainiae extract) have related to the extraction solvent of Semen Lepidii extract (Semen Descurainiae extract) and effect but have not related to the concrete effective site of Semen Lepidii (Semen Descurainiae) antitumor action and the name of an article in detail preparing application > > in antitumor drug.
summary of the invention
One of object of the present invention is that lepidii,semen (stir-fry) anti-cancer effective component extract and preparation method thereof is provided.
Two of object of the present invention is to provide said active component extract in the application of preparing in antitumor and related drugs.
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 water-saturated n-butanol position, cross silicagel column, with methylene chloride-methanol system, carry out gradient elution, collect the eluent that dichloromethane and methanol volume ratio are 5:1, boil off the lepidii,semen anti-cancer effective component extract that solvent obtains.
A method of preparing above-mentioned lepidii,semen anti-cancer effective component extract, it comprises the steps:
Step 1. is pulverized the lepidii,semen of parch, and water reflux, extract, is cooling, centrifugal, and concentrated extracting solution carries out precipitate with ethanol with 95% ethanol, and the centrifugal precipitation of removing, concentrates and remove ethanol, obtains extracting solution;
Step 2. extracts the extracting solution of step 1 gained successively with ethyl acetate, water saturated butanol solution, collect 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 reclaims after organic solvent, obtains lepidii,semen anti-cancer effective component extract.
The present invention further provides lepidii,semen anti-cancer effective component extract in the application of preparing in antitumor drug.To suppress U251, HepG 2, SGC, mcf-7, SCLC multiplication capacity be that activity index carries out anti-tumor activity experiment.Experimental result shows, in cellulotoxic experiment, can obviously suppress U251, HepG in vitro 2, SGC, mcf-7, SCLC propagation, there is cytotoxicity, can be used for preparing antineoplaston medicine.
beneficial effect
1, advantage of the present invention and effect are to adopt the conventional methods such as solvent extraction, extraction, from lepidii,semen, extract 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 expanded 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
Listed embodiment contributes to those skilled in the art to understand better the present invention below, but does 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, with the water reflux, extract, 3h of 10 times of quality, residue after centrifugal repeats to extract 1 time, centrifugal, merge all aqueous extracts, concentrated extracting solution to crude drug mass number (gram) 80% volume (ml), add 95% ethanol and carry out precipitate with ethanol, in medicinal liquid, the concentration of ethanol is about 60% ~ 70%, hold over night, centrifugal, discard precipitation, decompression recycling ethanol, adding isopyknic ethyl acetate is extracted to organic facies and approaches colourless, water saturated n-butanol extraction 6 times for remaining water liquid, reclaim n-butyl alcohol, obtain water-saturated n-butanol extract.Get the saturated n-butanol extract of suitable quantity of water and cross silicagel column, with methylene chloride-methanol (100:0,50:1, the 20:1 of different volumes ratio, 10:1,5:1,1:1 and 0:100) carry out gradient elution, each gradient effluent volume used is 4 times of column volumes, collected volume is than the methylene chloride-methanol eluent for 5:1, concentrated, vacuum drying, obtains 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 Shimadzu shimpack C18 post (4.6 × 150mm), and flow velocity is 1.0ml/min.Gradient elution: A phase: 0.01% formic acid-water; B phase: methanol, elution program is 0.0min~45.00min:B:2%~50%.Lepidii,semen anti-cancer effective component extract, under this chromatographic condition, mainly occurs that 5 retain peak, 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, with the water reflux, extract, 2h of 8 times of quality, residue after centrifugal repeats to extract 2 times, centrifugal, merge all aqueous extracts, concentrated extracting solution to crude drug mass number (gram) volume (ml), add 95% ethanol and carry out precipitate with ethanol, in medicinal liquid, the concentration of ethanol is about 60%, hold over night, centrifugal, discard precipitation, decompression recycling ethanol, adds isopyknic ethyl acetate and is extracted to organic facies and approaches colourless, remaining water liquid approaches colourless by n-butanol extraction to organic facies, collect n-butanol portion.Get appropriate 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, each gradient effluent volume used is 4 times of column volumes, collect the eluent of 5:1, concentrated, vacuum drying, obtains 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.Result shows, the reservation situation of lepidii,semen anti-cancer effective component extract in 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, with the water reflux, extract, 2h of 8 times of quality, residue after centrifugal repeats to extract 2 times, centrifugal, merge all aqueous extracts, concentrated extracting solution to crude drug mass number (gram) volume (ml), add 95% ethanol and carry out precipitate with ethanol, in medicinal liquid, the concentration of ethanol is about 60%, hold over night, centrifugal, discard precipitation, decompression recycling ethanol, adds isopyknic ethyl acetate and is extracted to organic facies and approaches colourless, remaining water liquid approaches colourless by n-butanol extraction to organic facies, collect n-butanol portion.Get appropriate 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, each gradient effluent volume used is 4 times of column volumes, collect the eluent of 5:1, concentrated, vacuum drying, obtains 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.Result shows, the reservation situation of lepidii,semen anti-cancer effective component extract in high performance liquid chromatography is consistent with embodiment's 1.
embodiment 4. anti-tumor activity experiments
The lepidii,semen effective site obtaining take embodiment 1 is tested medicine, In vitro culture U251, HepG 2, SGC, mcf-7, SCLC, get the cell of exponential phase of growth, with after trypsinization, 1500r ﹒ min -1centrifugal 5min, is resuspended in cell in culture medium, and cell concentration is adjusted to 2 × 10 4individual/ml, is inoculated in 96 porocyte culture plates, every hole 100 μ l, 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, all establishes 3 multiple holes for each group.After dosing 48h, every hole adds 5mg ﹒ ml -1mTT reagent 20 μ l, continue to be placed in 37 ℃ and cultivate 4h.Every hole adds DMSO100 μ l, and vibration mixes, and measures the absorbance of each hole at wavelength 570nm place.Calculate cell proliferation inhibition rate: suppression ratio (%)=(1-experimental group absorbance/matched group absorbance) × 100%.
The inhibitory action of table 1 lepidii,semen extract to U251
Figure 273399DEST_PATH_IMAGE001
The suppression ratio (%) of table 2 lepidii,semen extract to human tumor cells
Tested material Concentration (μ g/ml) HepG 2 mcf-7 SCLC SGC
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 obtaining with embodiment 2-3 repeats the present embodiment and 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 water-saturated n-butanol position, cross silicagel column, with methylene chloride-methanol system, carry out gradient elution, collect the eluent that dichloromethane and methanol volume ratio are 5:1, boil off solvent, obtain lepidii,semen anti-cancer effective component extract.
2. prepare a method for above-mentioned lepidii,semen anti-cancer effective component extract, it is characterized in that it comprises the steps:
Step 1. is pulverized the lepidii,semen of parch, and water reflux, extract, is cooling, centrifugal, and concentrated extracting solution carries out precipitate with ethanol with 95% ethanol, and the centrifugal precipitation of removing, concentrates and remove ethanol, obtains extracting solution;
Step 2. extracts the extracting solution of step 1 gained successively with ethyl acetate, water saturated butanol solution, collect 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 reclaims after organic solvent, obtains lepidii,semen anti-tumor effective component extract.
3. lepidii,semen anti-cancer effective component extract according to claim 1 is in the application of preparing in 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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