CN103027931B - Hydnocarpus hainanensis anti-tumor effective component extractive as well as preparation method and application thereof - Google Patents
Hydnocarpus hainanensis anti-tumor effective component extractive as well as preparation method and application thereof Download PDFInfo
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Abstract
The invention provides a hydnocarpus hainanensis anti-tumor effective component extractive, which is obtained by the following steps of: crushing hydnocarpus hainanensis fruits and extracting under heating by utilizing 70-95% ethanol; distilling an extracted solution to recycle ethanol; adding water and dispersing; extracting by ethyl acetate; separating an extractive by a silica gel column; and eluting by using different ratios of dichloromethane and methanol To obtain the thehydnocarpus hainanensis anti-tumor effective component extractive when the ratio of dichloromethane to methanol is 20: 1. The extractive disclosed by the invention has the effect of obviously inhibiting human glioma cells U251 and can be used for preparing an anti-tumor medicine. The invention discloses a preparation method of the anti-tumor effective component extractive.
Description
Technical field
The present invention relates to Hydnocarpus hainanensis (Merr.) Sleum. anti-cancer effective component extract, containing its pharmaceutical composition and method for making thereof and purposes.
Background technology
Tumor is one of most serious disease of harm humans health in the world today, the experts and scholars of countries in the world find method and the medicine for the treatment of tumor in active research, especially very active to chemosensitive test Quality Research in Chinese medicine, about the zoopery of this respect and Preliminary Clinical Observation show that in Chinese medicine, many kinds of substance has stronger inhibitory action to zoografting tumor, the immunologic function of energy enhancing body, alleviates the toxic reaction of radiotherapy, chemotherapy.
About there is 86 genus 850 kinds in the Flacourtiaceae plant whole world, and be mainly distributed in subtropical and tropical zones, only a few is extended down to Temperate Region in China, is distributed with 6 races 12 belongs to 45 kind of 6 mutation in China.The biological activity of Flacourtiaceae plant mainly contains anticancer and antiviral activity, water extract and ethanol extract also show good blood sugar lowering, blood fat reducing and neutralize Neurotoxic effect (Chai Xingyun, Lu Yanan, Ren Hongyan, Deng. CHINA JOURNAL OF CHINESE MATERIA MEDICA, 2006,31 (4): 269.).Semen Hydnocarpi is mainly distributed in Hainan and some areas of Guangxi in China, has effect of wind-damp dispelling, detoxicating and killing parasites, is clinically used for the diseases such as leprosy, rash tinea, syphilitic skin lesion.
India scholar Sharma DK etc. are separated earlier and obtain 3 flavanolignan's constituents: hydnowightin, hydnocarpin and neohydnocarpin from hydnocarpus wightiana Hydnocarpus wightiana.(Sharma DK, Ranganathan KR, Parthasarathy MR, et al.Plant Med, 1979,37 (1): 79) all there are the effect reducing S-TG in these three kinds of flavanolignans, and Hydnocarpin has obvious antiinflammatory action.In addition, these three kinds of compounds all have against murine class L-1210 leukemic lymphoblastoid, KB rhinopharyngeal neoplasm, adenocarcinoma of colon, HeLa-S
3, and osteosarcomatous effect.Three kinds all can not be suppressed lung carcinoma cell.Hydnocarpin can suppress neuroglial cytoma.Hydnocarpin and neohydnocarpin can suppress Tmolt
3leukemia.
Chinese patent application CN03138732.2 " a kind of medicine of Therapeutic cancer ", CN200810146656.9 " a kind of medicine of Therapeutic cancer ", CN200910143487.8 " Chinese medicine preparation for the treatment of cervical cancer ", CN200910134392.X " treatment tumor prescription " relate to the common antitumor action that Semen Hydnocarpi combines other Chinese medicines, but all using Semen Hydnocarpi as certain Herba indigoferae Pseudotinctoriae in Chinese medicine prescription, do not relate to Semen Hydnocarpi self antitumor active site.
Summary of the invention
An object of the present invention is, provides a kind of Hydnocarpus hainanensis (Merr.) Sleum. anti-cancer effective component extract and preparation method thereof.
Two of object of the present invention is, provides described active component extract preparing the application in antitumor and related drugs thereof.
Technical scheme of the present invention is as follows:
A kind of Hydnocarpus hainanensis (Merr.) Sleum. anti-cancer effective component extract, it is oil removing after being pulverized by Hydnocarpus hainanensis (Merr.) Sleum. fruit, extract with the alcohol heating reflux of 70%-95%, extracting solution Distillation recovery ethanol, then add aqueous dispersion, be extracted with ethyl acetate, extract crosses silicagel column, dichloromethane in varing proportions and methanol mixed solvent eluting, obtain Hydnocarpus hainanensis (Merr.) Sleum. anti-cancer effective component extract at the eluent of methylene chloride-methanol ratio 20: 1 part through boiling off solvent successively.This position has the effect significantly suppressing human glioma cell U251, may be used for preparing antitumor drug.
Prepare a method for above-mentioned Hydnocarpus hainanensis (Merr.) Sleum. anti-cancer effective component extract, it comprises the steps:
Hydnocarpus hainanensis (Merr.) Sleum. fruit is pulverized rear oil removing by step 1.;
The Semen Hydnocarpi medical material of step 1 gained extracts with the alcohol heating reflux of 70%-95% by step 2., cooling, and filter, concentrated extracting solution obtains extractum;
The extractum of step 2 is added aqueous dispersion by step 3., with extraction into ethyl acetate, after extract distillating recovering solvent, obtains acetic acid ethyl ester extract;
The acetic acid ethyl ester extract of step 3 is crossed silicagel column by step 4., dichloromethane in varing proportions and methanol mixed solvent eluting.Be that the eluent of 20: 1 parts obtains Hydnocarpus hainanensis (Merr.) Sleum. anti-cancer effective component extract through boiling off solvent in methylene chloride-methanol ratio.
The above-mentioned method preparing Hydnocarpus hainanensis (Merr.) Sleum. anti-cancer effective component extract, the oil removing described in step 1 can be sarcocarp squeezing oil removing or the oil removing of sarcocarp Petroleum ether extraction.
The above-mentioned method preparing Hydnocarpus hainanensis (Merr.) Sleum. anti-cancer effective component extract, described sarcocarp Petroleum ether extraction oil removing uses petroleum ether extraction after step 2 gained extractum is added aqueous dispersion.
Invention further provides Hydnocarpus hainanensis (Merr.) Sleum. anti-tumor effective component and prepare the application in antitumor drug.Anti-tumor activity experiment is carried out as activity index to suppress the competence for added value of human glioma cell U251.Experimental result shows, in vitro in cytotoxicity test, obviously can suppress the increment of U251, have cytotoxicity, can be used for preparing antineoplaston medicine.
Beneficial effect
(1) advantage of the present invention and effect are to adopt solvent extraction, extraction, the conventional methods such as silicagel column, extract and obtain the active component with antitumor action from Hydnocarpus hainanensis (Merr.) Sleum..This active component the experiment proved that the activity having vitro inhibition human glioma cell U251 and grow.
(2) the present invention is using Hydnocarpus hainanensis (Merr.) Sleum. as antitumor drug, specific to its antineoplastic active site, establishes thinking, for the modernization of Chinese medicine plays impetus for better studying its antitumor mechanism.
Detailed description of the invention
Listed embodiment contributes to those skilled in the art and understands the present invention better below, but does not limit the present invention in any way.
The preparation of embodiment 1. Hydnocarpus hainanensis (Merr.) Sleum. anti-cancer effective component extract
Shelled by Hydnocarpus hainanensis (Merr.) Sleum. fruit 5kg, sarcocarp deoils with squeezer, and shell is pulverized afterwards and after deoiling, medicinal residues merge.With 95% alcohol reflux 2 hours, amount of alcohol is 3 times of Semen Hydnocarpi quality, and the residue after filtration repeats extraction 1 time, filters, merge twice ethanol extract 30L, reclaim ethanol extremely near without alcohol taste, continue to be concentrated into 600ml, add aqueous dispersion, add isopyknic extraction into ethyl acetate 4 times, combined ethyl acetate extract 2400ml, reclaims ethyl acetate, about obtains ethyl acetate extract and is about 100g.Get 20g ethyl acetate extract, cross silicagel column, carry out eluting with dichloromethane, methylene chloride-methanol (100: 1,50: 1,20: 1) as eluant successively.Dry to 60 DEG C of vacuum drying ovens after each eluent concentrating under reduced pressure, obtain methylene chloride-methanol ratio be 20: 1 elution fraction be Semen Hydnocarpi anti-cancer effective component extract.
The preparation of embodiment 2. Hydnocarpus hainanensis (Merr.) Sleum. anti-cancer effective component extract
Pulverized by Hydnocarpus hainanensis (Merr.) Sleum. fruit 5kg, with petroleum ether (60-90 DEG C) merceration 2 hours, petroleum ether consumption was 2 times of Semen Hydnocarpi quality.Take off a layer medicinal residues, again with petroleum ether merceration 2 hours.Lower floor's medicinal residues 80% alcohol reflux 3 hours, amount of alcohol is 3 times of Semen Hydnocarpi quality, and the residue after filtration repeats extraction 1 time, filters, merge twice ethanol extract, reclaim ethanol extremely near without alcohol taste, continue to be concentrated into 600ml, add aqueous dispersion, add isopyknic extraction into ethyl acetate 4 times, combined ethyl acetate extract 2400ml, reclaims ethyl acetate, about obtains ethyl acetate extract and is about 100g.Get 20g ethyl acetate extract, cross silicagel column, carry out eluting with dichloromethane, methylene chloride-methanol (100: 1,50: 1,20: 1) as eluant successively.Dry to 60 DEG C of vacuum drying ovens after each eluent concentrating under reduced pressure, obtain methylene chloride-methanol ratio be 20: 1 elution fraction be Semen Hydnocarpi anti-cancer effective component extract.
Hydnocarpus hainanensis (Merr.) Sleum. anti-tumor effective component extract is through the further separation and purification of liquid phantom preparing chromatogram and NMR carbon spectrum hydrogen analysis of spectrum, and preliminary judgement is mainly containing hydnocarpin and other unknown materials.
The preparation of embodiment 3. Hydnocarpus hainanensis (Merr.) Sleum. anti-cancer effective component extract
Hydnocarpus hainanensis (Merr.) Sleum. fruit 5kg is pulverized, with 95% alcohol reflux 2 hours, amount of alcohol is 3 times of Semen Hydnocarpi quality, residue after filtration repeats extraction 1 time, filters, merges twice ethanol extract, reclaim ethanol near without alcohol taste, continue to be concentrated into 600ml, add aqueous dispersion, add isopyknic petroleum ether extraction 3 times.Water layer adds isopyknic extraction into ethyl acetate 6 times, combined ethyl acetate extract, reclaims ethyl acetate, about obtains ethyl acetate extract and is about 100g.Get 20g ethyl acetate extract, cross silicagel column, carry out eluting with dichloromethane, methylene chloride-methanol (100: 1,50: 1,20: 1) as eluant successively.Dry to 60 DEG C of vacuum drying ovens after each eluent concentrating under reduced pressure, obtain methylene chloride-methanol ratio be 20: 1 elution fraction be Semen Hydnocarpi anti-cancer effective component extract.
The preparation of embodiment 4. Hydnocarpus hainanensis (Merr.) Sleum. anti-tumor effective component extract
Pulverized by Hydnocarpus hainanensis (Merr.) Sleum. fruit 100g, with petroleum ether merceration 2 hours, petroleum ether amount was 2 times of Semen Hydnocarpi amount.Take off a layer medicinal residues, again with petroleum ether merceration 2 hours.Lower floor's medicinal residues 95% alcohol reflux 2 hours, amount of alcohol is 3 times of Semen Hydnocarpi quality, and the residue after filtration repeats extraction 1 time, filter, merge twice ethanol extract, reclaim ethanol near without alcohol taste, continue to be concentrated into 50ml, add aqueous dispersion, add isopyknic petroleum ether extraction 2 times.Add isopyknic extraction into ethyl acetate 4 times, combined ethyl acetate extract, reclaim ethyl acetate, about obtain ethyl acetate extract and be about 10g.Get 2g ethyl acetate extract, cross silicagel column, carry out eluting with dichloromethane, methylene chloride-methanol (100: 1,50: 1,20: 1) as eluant successively.Dry to 60 DEG C of vacuum drying ovens after each eluent concentrating under reduced pressure, obtain methylene chloride-methanol ratio be 20: 1 elution fraction be Semen Hydnocarpi anti-cancer effective component extract.
Hydnocarpus hainanensis (Merr.) Sleum. anti-cancer effective component extract is through the further separation and purification of liquid phantom preparing chromatogram and NMR carbon spectrum hydrogen analysis of spectrum, and preliminary judgement is mainly containing hydnocarpin and other unknown materials.
Embodiment 5. anti-tumor activity is tested
The Hydnocarpus hainanensis (Merr.) Sleum. anti-cancer effective component extract obtained with embodiment 1 is test medicine.
In vitro culture human glioma cell U251, gets the cell of exponential phase of growth, and after trypsinization, the centrifugal 5min of 1500r/min, be resuspended in by cell in culture medium, cell concentration is adjusted to 2*10
4individual/ml, is inoculated in 96 porocyte culture plates, every hole 100 μ l, and imitated cell culture incubator (37 DEG C, 5%CO
2) in cultivate 24 hours.Add test medicine 20 μ l afterwards, it is 0.5%DMSO that negative control group adds final concentration, all establishes 3 multiple holes for each group.Respectively dosing 24 hours, 48 hours, 72 is constantly little, and every hole adds 5mg/mlMTT reagent 20 μ l, continues to be placed in 37 DEG C and cultivate 4 hours.Every hole adds DMSO100 μ l, and vibration mixing, 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%.
Table 1 Hydnocarpus hainanensis (Merr.) Sleum. extract anti-tumor activity experiment (U251)
Claims (5)
1. a Hydnocarpus hainanensis (Merr.) Sleum. anti-cancer effective component extract, it is characterized in that: it is oil removing after being pulverized by Hydnocarpus hainanensis (Merr.) Sleum. fruit, extract with the alcohol heating reflux of 70%-95%, extracting solution Distillation recovery ethanol, then add aqueous dispersion, be extracted with ethyl acetate, extract crosses silicagel column, dichloromethane in varing proportions and methanol mixed solvent eluting, obtain Hydnocarpus hainanensis (Merr.) Sleum. anti-cancer effective component extract at the eluent of methylene chloride-methanol ratio 20: 1 part through boiling off solvent successively.
2. prepare a method for Hydnocarpus hainanensis (Merr.) Sleum. anti-cancer effective component extract described in claim 1, it is characterized in that it comprises the steps:
Hydnocarpus hainanensis (Merr.) Sleum. fruit is pulverized rear oil removing by step 1.;
The Semen Hydnocarpi medical material of step 1 gained extracts with the alcohol heating reflux of 70%-95% by step 2., cooling, and filter, concentrated extracting solution obtains extractum;
The extractum of step 2 is added aqueous dispersion by step 3., with extraction into ethyl acetate, after extract distillating recovering solvent, obtains acetic acid ethyl ester extract;
The acetic acid ethyl ester extract of step 3 is crossed silicagel column by step 4., dichloromethane in varing proportions and methanol mixed solvent eluting are that the eluent of 20: 1 parts obtains Hydnocarpus hainanensis (Merr.) Sleum. anti-cancer effective component extract through boiling off solvent in methylene chloride-methanol ratio.
3. according to the method preparing Hydnocarpus hainanensis (Merr.) Sleum. anti-cancer effective component extract described in claim 2, it is characterized in that: the oil removing described in step 1 is sarcocarp squeezing oil removing or the oil removing of sarcocarp Petroleum ether extraction.
4. the Hydnocarpus hainanensis (Merr.) Sleum. anti-cancer effective component extract described in claim 1 is preparing the application in antitumor drug.
5. preparing the application in antitumor drug according to the Hydnocarpus hainanensis (Merr.) Sleum. anti-cancer effective component extract described in claim 4, it is characterized in that: described antitumor drug is being anti human nerve glioma cell of U251 medicine.
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